Replicon RNA vaccines: design, delivery, and immunogenicity in infectious diseases and cancer

Abstract

Replicon RNA (RepRNA) represents a cutting-edge technology in the field of vaccinology, fundamentally transforming vaccine design and development. This innovative approach facilitates the induction of robust immune responses against a range of infectious diseases and cancers. RepRNA vaccines leverage the inherent capabilities of RNA-dependent RNA polymerase associated with self-replicating repRNA, allowing for extreme replication within host cells. This process enhances antigen production and subsequently stimulates adaptive immunity. Additionally, the generation of double-stranded RNA during RNA replication can activate innate immune responses. Numerous studies have demonstrated that repRNA vaccines elicit potent humoral and cellular immune responses that are broader and more durable than those generated by conventional mRNA vaccines. These significant immune responses have been shown to provide protection in various models for infectious diseases and cancers. This article will explore the design and delivery of RepRNA vaccines, the mechanisms of immune activation, preclinical studies addressing infectious diseases and tumors, and related clinical trials that focus on safety and immunogenicity.

Background

Replicon RNA (repRNA) has received public attention since the COVID-19 pandemic due to the urgent need to develop an effective vaccine. In the face of major epidemics, replicon vaccines have already been put into emergency use, including a VSV-based Ebola virus (EBOV) vaccine, ERVEBO®, approved by the US Food and Drug Administration (FDA) [1] and GEMCOVAC-OM approved in India [2]. RNA replicons derived from positive sense/negative sense, single-stranded RNA viruses are deployed for vaccine development due to the RNA-dependent RNA polymerase (RdRp) activity, which induces approximately 10⁶ copies per cell after the subgenomic RNA translocates into the cytoplasm [3]. Alphaviruses, flaviviruses, Nodamura virus (NoV) (positive-strand RNA virus), Paramyxoviruses, and rhabdoviruses (negative-strand RNA virus) are applied for repRNA vaccine vectors [4, 5]. By replacing the structural genes of the viral genome with the gene of interest (GOI), and preserving the non-structural genes that encode RdRp, repRNAs can induce massive RNA amplification and protein expression when used at low doses [6]. The absence of structural proteins prevents the production of virus progeny. Instead, the self-replication mechanism of replicons generates numerous RNA copies in the cytoplasm, elevating the expression of antigens [7]. The majority of repRNA vaccine studies involve replicons with a single mRNA transcript. In contrast, some studies use a two-helper system [8], in which alphavirus non-structural protein 1–4 (nsP1–4) genes and the GOI are encoded in the expressing vector, and structural protein genes are expressed in the helper vector. The transfection of both vectors to mammalian cells gives rise to replication-deficient alphavirus particles [9]. The RNA replication process generates double-stranded RNA (dsRNA) and long RNA, which in both systems stimulate innate immune responses as an immunological adjuvant [8].

Nucleic acid vaccines have gained attention due to capabilities of enhancing prophylactic and therapeutic efficacy, and responding rapidly against emergent infectious disease [10, 11]. Among different nucleic acids, DNA was first applied in vaccine manufacturing due to the stability, and large-scale production [12]. However, the efforts shifted to RNA vaccinology since the poor outcomes of DNA vaccines in human clinical trials [13]. RNA vaccines' cytosolic location and transient properties guarantee higher safety levels than DNA vaccines, which promote protein expression dependent on nuclear delivery and promoter expression, and risk genome integration [10]. Commonly seen RNA vaccinology technologies include mRNA vaccines and repRNA vaccines. The antigen expression level induced by mRNA vaccine is proportionate to the number of mRNA transcripts delivered to the host cell. Evoking sufficient immune response requires adequate antigen expression, which demands large doses or repeat administration of mRNA vaccine [10]. However, one repRNA molecule leads to multiple copies of the encoded protein and amplified, prolonged antigen expression that lasts several weeks to months after a single injection [14]. A clinical trial has revealed that a repRNA vaccine achieved comparable immunogenicity at one-tenth the dose of an equivalent mRNA vaccine. However, the incidence of side effects was similar across the treatment groups [15]. Nonetheless, repRNA being a special type of mRNA, the transient nature of mRNA brings about the necessity of repeated administration. Moreover, repRNA vaccines have superior self-adjuvanting properties than mRNA vaccines due to the activation of innate immune pathways by components [16]. Additionally, more robust cell-mediated immunity and stronger CD4⁺ T cell and CD8⁺ T cell responses are witnessed in repRNA-treated subjects than mRNA-treated ones, making repRNA-based vaccines more suitable for cancer therapy [17]. Both mRNA vaccine and repRNA vaccine require ultra-low storage temperature [18].

Conventional delivery of heterogeneous genes in studies is carried out by viral vectors derived from DNA viruses or retroviruses, including Adenovirus, Lentivirus, Herpesvirus, and Adeno-associated viruses [19,20,21,22,23]. Lentivirus and adeno-associated viruses are attributed to the long-term expression of GOI by integration into the host genome [24] or the form of episomal DNA [25], respectively. Adenovirus [26] and herpesvirus [23] induce transient GOI expression. However, the application of viral vectors is limited due to the tropism and high prevalence of similar viruses, which impair the efficacy. Chikungunya virus (CHIKV) is a mosquito-borne alphavirus [27] that has been applied in repRNA vaccine studies [28, 29] and has wide tropism due to its access to multiple receptors and cellular factors, such as Actin gamma 1, collagen type I-alpha-2, and protein tyrosine phosphatases non-receptor type 2, prohibitin 1, matrix remodeling associated 8, dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing non-integrin, T-cell immunoglobulin and mucin structural domain 1, and glycosaminoglycans [30,31,32,33,34,35]. However, CHIKV has taken outbreaks in La Reunion, Africa, Asia, and South America, resulting in concerns about the application as vaccine vectors in these areas [36]. In this case, different strains of alphavirus can be applied. Since different tropisms are detected in Old and New-World alphavirus, which gives rise to the theory that exchanging the glycoprotein between Old and New-World alphavirus can modify the tropism of the viral vector [37]. The seroprevalence against CHIKV in the human population in non-tropical climates is lower than other viruses. Under the circumstance that the seroprevalence against a particular alphavirus is high, the change of glycoproteins for a low seroprevalence alphavirus can settle the problem [29, 38].

Challenges have emerged as the investigation focusing on the repRNA vaccine goes deeper. Firstly, repRNAs are RNase-sensitive and inefficiently taken by DCs [39]. Nano-delivery systems, including lipid nanoparticles (LNPs), liposomes, lipid polycomplexes, polymer materials, micelles, and polypeptides, that wrap and protect repRNA help repRNA transfect immune cells without unwanted toxicity or immunogenicity [40]. Secondly, in vivo, innate immune responses elicited by repRNA limit transgene expression and induce dose-limiting reactogenicity. Intramuscular administration of localizing cationic nanocarrier formulation (LION) delivered repRNA resulted in localized biodistribution that triggered local instead of systemic inflammatory responses [41]. A temperature-sensitive repRNA vaccine that is inactivated at core temperature has been constructed, which also restricts systemic inflammation [42]. Thirdly, ultra-low temperature (−80 to −20 °C) storage conditions hamper the transportation to and application of repRNA vaccines in remote areas. A thermostable lyophilized repRNA vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was developed that can be stored at 2–8 °C for one year, or 25 °C for one week [43]. Another lyophilized mRNA-LNP vaccine constructed by Ai et al. can retain the physiochemical properties and bioactivities at 25 °C over six months [44]. Moreover, the wide variety of viral vector options, including flaviviruses, alphaviruses, NoV, etc., needs to be researched [17]. Furthermore, high construction costs, the requirements for favorable pharmacological profiles, and the lack of cell targeting restrict the development and application of repRNA vaccines [39].

We conducted a comprehensive review of the theoretical underpinnings of replicon vaccines, as well as the preclinical studies and clinical trials associated with them. Replicon vaccines hold significant promise for transforming vaccine development due to their capacity for rapid production and high expression of the GOI at low doses. However, their mechanisms remain not fully understood and warrant further investigation.

RepRNA vaccine viral vectors

Alphavirus

Alphaviruses are a group of single-strand, positive-sense RNA viruses [45]. Alphaviruses are one of the most common genera utilized as the backbone to construct repRNA vaccines in both clinical studies and preclinical studies, including Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), Sindbis virus (SINV), CHIKV [29, 46,47,48,49], eastern equine encephalitis virus (EEEV) [50], Mayaro virus [51], Ross River virus (RRV) [52], and western equine encephalitis virus (WEEV) [53]. The infection of alphaviruses induces 10⁶ copies of subgenomic RNA in the host cytoplasm per cell [3]. 240 capsid protein monomers formed nucleocapsid, one copy of gRNA, a lipid bilayer, and 80 trimers of heterodimers of the glycoproteins E1/E2 make up a single virion. Its gRNA (11.5 kb) consists of 5’ and 3’UTR, an integrated region, and 2 open reading frames (ORF1 and ORF2). 4 nonstructural proteins (nsP1, 2, 3, 4) are encoded in the ORF1 that participate in the assembly of RdRp (Fig. 1). The ORF2 sequence expresses structural proteins C, E3, E2, E1, TF, and 6 K [54, 55]. GOI sequences can replace these structural proteins, inducing the formation of replicons that are self-replicative but defective in the production of viral particles [29]. Alphaviruses enter host cells through clathrin-mediated endocytosis. Once membrane fusion occurs in endosomes, the virus particles disassemble, releasing gRNA into the cytoplasm of host cells. Subsequently, gRNA is translated into nsPs, and structural polyproteins, which facilitate the synthesis of negative- (-ssRNA) and positive-strand RNA (+ ssRNA) and support viral replication. New virions are assembled and released through budding from the host cell plasma membrane (Fig. 2) [56].

Fig. 1

Illustration of the genome composition of viruses applied as viral vectors for vaccine design. Positive sense ssRNA viruses: alphavirus, flavivirus, Nodamura virus, and negative sense ssRNA viruses: paramyxovirus, rhabdovirus are applied to design vaccines. C/CP, capsid protein; prM, pre-membrane protein; E, envelope protein; N, nucleocapsid protein; P, phosphoprotein; M, matrix protein; F, fusion glycoprotein; G, glycoprotein; L, large polymerase component; H, hemagglutinin glycoprotein

Fig. 2

Illustration of the life cycle of alphaviruses. Alphaviruses initiate infection by binding to attachment factors (e.g., heparan sulfate) and specific receptors (e.g., MXRA8, LDLRAD3, VLDLR, and ApoER2) on the cell surface. Viral entry is primarily mediated through clathrin-mediated endocytosis. As the endosome traffics within the cell, its acidic environment induces conformational changes in the viral envelope proteins, facilitating fusion with the endosomal membrane. This allows the virus to enter the cytoplasm, where nucleocapsid disassembly occurs, after which the incoming + ssRNA is translated into P123 and P1234 polyproteins. Then nsP2 protease cleaves P1234 polyprotein in cis, dissociating nsP4, which together with P123 forms the early replicase complex that synthesizes -ssRNA. Genomic and subgenomic RNAs are synthesized using the -ssRNA as a template. Structural polyproteins, C-pE2-6 K/TF-E1, are translated from subgenomic RNA. Afterward, the early complex is processed into a transient nsP1, P23, and nsP4 complex, and eventually into the late replicase containing nsP1, nsP2, nsP3, and nsP4. The E2 envelope glycoprotein is synthesized and covalently bonded to E3, forming the polyprotein pE2 that interacts with E1. The TF protein is generated through a ribosomal frameshift event during the translation of the 6 K gene. Meanwhile, the viral C protein is released via its autoprotease activity and binds to the newly synthesized genomic RNA, assembling into the nucleocapsid. The remaining structural polyprotein undergoes processing and maturation in the endoplasmic reticulum, where host signal peptidases cleave pE2, 6 K/TF, and E1. In the Golgi network, furin-like proteases (FLP) cleave pE2 into the individual envelope glycoproteins E2 and E3. For some alphaviruses (e.g., SINV, SFV, and VEEV), E3 may remain associated with the virion. The mature E2-E1 glycoproteins are then transported to the plasma membrane, where they associate with the nucleocapsid to facilitate budding and release of the mature virions

Flavivirus

Genera Flavivirus is a group of single-stranded, positive-sense RNA viruses. Kunjin virus (KUNV) [57,58,59,60], West Nile virus (WNV) [61], yellow fever virus (YFV) [62, 63], dengue virus (DENV) [64,65,66], tick-borne encephalitis virus (TBEV) [67,68,69], and Zika virus (ZIKV) [70] etc. are developed as backbone of repRNA vaccines. The viral shell consists of 180 copies of glycosylated E and M proteins. Nucleocapsid complexes, containing 1 copy of viral genome RNA and multiple copies of capsid protein, form the core of virion. The viral genome includes a capped 5’ UTR but lacks a poly(A) end, a 3’ UTR, and one ORF, which encodes 3 structural proteins (capsid, C; precursor membrane protein, prM; envelope, E), and 7 nonstructural proteins (nsP1, nsP2A/2B, nsP3, nsP4A/4B and nsP5) (Fig. 1) [71,72,73]. The ORF encodes a polyprotein in an NH2-C-prM(M)-E-nsP1-nsP2A-nsP2B-nsP3-nsP4A-2K-nsP4B-nsP5-COOH manner [73, 74]. By attaching the viral E proteins to cellular receptors, flaviviruses are internalized into host cells by clathrin-mediated endocytosis. For DENVs, DC-specific intracellular adhesion molecule-3-grabbing non-integrin, heparan sulfate, mannose receptors, heat-shock proteins (HSP) 70 and 90 and immunomodulatory proteins (TIM/TAM receptors) can mediate the entry into host cells. In endosomal compartments, the viral envelope fuses with the endosomal membrane in response to the low pH environment, releasing the viral RNA genome into the cytoplasm. The viral RNAs are further translated into polyproteins by ribosomes of host cells, which are processed into structural and non-structural proteins within the endoplasmic reticulum (ER) membrane. Non-structural proteins form replication complexes on the ER membrane, where viral RNA replication occurs. The newly synthesized RNA is encapsidated and buds into the ER lumen, forming immature virions, which are then transported for maturation via the GBF1-dependent host secretory pathway or alternatively through secretory autophagy. Finally, mature virions are released as free virions or in membrane-enclosed forms derived from autophagosomes (Fig. 3) [75, 76].

Fig. 3

Illustration of the life cycle of flaviviruses. The flaviviruses infect host cells starting with the attachment of viral E protein with cellular receptors, packaged into clathrin-coated vesicles. Subsequently, the mild acidic environment in endosomal compartments induces the fusion of the viral envelope and endosomal membrane, triggering the translocation of the nucleocapsid into the cytoplasm, where the viral RNA is translated into a polyprotein and inserted into the ER. The polyprotein is further processed by protease, generating three structural proteins and at least seven nsPs. Viral replication complexes are assembled from nsPs. Besides, the viral proteins facilitate the formation of replication organelles (ROs) and vesicle packets (VPs) within the ER, shielding the virus from the cytoplasm. ROs are the assembly site, where + ssRNAs are synthesized and transported to VPs. Capsid proteins on lipid droplets and heterodimers of prM and E proteins are recruited to VPs along with + ssRNAs for encapsidation. Then VPs bud into the ER, producing immature virions, which accumulate within ER cisternae and are subsequently transported for maturation via either the GBF1-dependent host secretory pathway or through secretory autophagy. Mature viruses can then be released as free virions or in membrane-enclosed forms derived from autophagosomes

Nodamura virus

Nodamura virus is a positive-sense, single-stranded, segmented RNA virus, the genome expressing two RNA molecules, RNA1 and RNA2. RNA1 encodes RdRp, while the RNA2 encodes viral capsid protein [77] (Fig. 1). The NoV replicon has a deletion of RNA2 and the GOI is inserted at the end of the RdRp ORF, separated from the RdRp gene by a T2A ribosome-shifting sequence [78]. Compared to the alphavirus replicon, the NoV replicon is short and capable of in vitro assembly into spherical virus-like particles (VLPs) with capsid protein from bromoviruses [79].

Paramyxovirus

Paramyxoviruses are a group of negative-sense, single-stranded RNA virus, which composes a non-segmented genome that encodes 6 proteins: the nucleoprotein (N), the phosphoprotein (P), matrix protein(M), fusion glycoprotein (F), hemagglutinin glycoprotein (H), and the large polymerase component (L) [80] (Fig. 1). The N protein, the most abundant viral protein, surrounds the RNA in a helical nucleocapsid structure, which is required for replication and transcription, and the formation of ribonucleoprotein in conjugation with the P and L proteins. The P gene encodes at least 3 gene products: the P, C, and V proteins. The M protein, and the H, and F glycoproteins constitute the envelop of the paramyxovirus virus. The M protein is associated with both the inner viral membrane and cytoplasmic domains of glycoproteins, which are speculated to stabilize or organize the membrane environment before budding. The H protein is regarded to interact with the host cell surface upon the attachment phase of infection. The H and F proteins permit viral entry by promoting membrane fusion. The F protein contributes to the erythrocyte hemolysis. The measles virus (MV) and the Newcastle disease virus (NDV) are applied as the repRNA vaccine viral vectors [81,82,83]. In the host cell's cytoplasm infected with the paramyxovirus, a complementary positive-sense RNA is synthesized from the negative-sense RNA template for translation and RNA replication [84]. Foreign genes can be inserted between the P and the M protein, or the H and the L protein [81].

Rhabdovirus

The Rhabdoviridae family comprises negative-sense single-stranded RNA viruses that are in the shape of a bullet. The genome consists of 5 genes: the nucleoprotein (N) gene, phosphoprotein (P) gene, matrix protein (M) gene, glycoprotein (G) gene, and large protein (L) gene that encodes for RdRp (Fig. 1) [85]. Rabies virus, from the genus Lyssavirus, and vesicular stomatitis virus (VSV) from the genus Vesiculovirus are subjected to vector engineering [86, 87]. The replication of Rhabdovirus RNA in the host cell cytoplasm resembles that of paramyxovirus.

Delivery forms and delivery carriers of repRNA vaccines

The delivery of repRNA vaccines generally takes four forms: 1. naked RNA, 2. DNA intermediates, 3. viral replicon particles (VRPs), and 4. synthetic replicons [8] (Fig. 4). Electroporation is normally used for the transfection of naked repRNA, which can be expensive and impractical for large-scale applications [29]. Replacing the RNA polymerase promoter in repRNA sequence with a mammalian host cell compatible eukaryotic RNA polymerase type II promoter such as CMV, DNA replicons can directly transfect mammalian cells. Once the DNA replicon is transported into the host cell, it is translocated into the nucleus, where the transcription happens, after which the mRNA is translocated to the cytoplasm for translation of the nsP1-4, forming the replicase complex that induces massive mRNA replication. The nuclear delivery of DNA replicon makes it less efficacious. Adding a nuclear localization signal in the vector potentiates the efficacy of DNA replicons [88]. VRPs use helper vectors that encode structural proteins that can assemble with repRNAs and conduct single-round infections [8]. VRPs exhibit strong lymphotropism, which endows them with the capacity to elicit and maintain robust immune responses [89, 90]. Furthermore, innate immune responses can be directly elicited by certain VRP. The H protein of the MV can directly activate TLR2 [91]. The G protein of VSV can activate immune responses associated with TLR4 [92]. However, the induction of immune responses targeting structural proteins may hamper the vaccine efficacy, which is evidenced by the production of antibodies that restrict secondary transduction in vivo and foster T-cell competition against the encoded antigens [8]. Moreover, the use of VRP induces risk in generating replication-competent viruses during manufacturing, which can be eliminated by splitting the helper RNA sequence in two, one encoding the capsid protein, and the other encoding the envelop glycoproteins [93]. Synthetic repRNA particles package repRNA in lipid formulations or nanoparticles [28, 94, 95], which is highly scalable, highly effective in cell transduction, and avoids the immune responses induced by VRPs [96,97,98].

Fig. 4

Schematic illustration of the delivery systems applied for replicon vaccine delivery. Six delivery systems are presented. RepRNAs are transfected to host cells through electroporation, VRPs, or synthetic replicon. Transfected RNAs localize in the cytoplasm, where RNA replication and translation are accomplished. DNA-launched replicons are delivered as naked plasmid DNA, LNP-encapsulated plasmid DNA, or Salmonella bactofection. Once the recombinant DNA is delivered to the host cells, it enters the nucleus and is transcribed into repRNA, which translocates to the cytoplasm and undergoes replication and translation. The synthetic replicon platforms are illustrated at the top of the figure

Different synthetic replicon delivery systems are being developed. Polyethyleneimine (PEI) polyplexes have proven to protect repRNA against degradation by nucleases and promote internalization by DCs [99, 100]. A coatsome-replicon vehicle based on Coatsome SS technologies achieved up to a ∼65-fold increase in the synthesis of the GOI encoded by RepRNA compared to conventional polyplexes [101]. Upon cellular uptake, ionizable polymeric micelles with phenylalanine moieties induced a pH-dependent membrane disruption, facilitating the delivery of RepRNA into the cytosol, and achieving more potent and sustained protein expression in vivo than other commercial transfecting reagents [102]. Nanolipoprotein particles formulated with cationic lipids showed potential in complexation with repRNA and improving GOI expression. The type and proportion of cationic lipids, and ratio of cationic nanolipoprotein particles to replicon and be further modified to achieve transfection efficiency [103]. Lipid inorganic nanoparticle (LION) complexes with repRNA through electrostatic interaction and maintains colloidal stability for a minimum of 3 months when stored at temperatures of 4 °C and 25 °C [104]. Chahal et al. developed a modified dendrimer nanoparticle delivery platform (MDNP), that utilizes modified dendrimers and lipid-anchored polyethylene glycol (PEG) to form stable nanoparticles that protect RNA from degradation by nucleases. MDNP has a large payload capacity, and a hexavalent vaccine was produced to express six Toxoplasma gondii-specific antigens. A single dose of the hexavalent vaccine generated complete protection against the Toxoplasma gondii challenge for more than six months [105]. Interestingly, a study developed a eukaryotic delivery approach to deliver DNA replicons with Salmonella bactofection, which enhances the delivery of repRNA into mammal cells [106].

The induction of immune responses of repRNA vaccines

Recombinant repRNA vaccines carrying antigen sequences or immunomodulatory sequences stimulate the host immune systems through different pathways, evoking innate and adaptive immunity responses (Fig. 5).

Fig. 5

Schematic illustration of immune responses induced by replicon vaccines. Innate immunity: the administration of recombinant replicon RNA induces the production of dsRNAs, which bind to different receptors in different intercellular locations: cytosolic dsRNA interacts with RIG-1 or MDA-5, endosomal dsRNA interacts with TLR3 or TLR7/8. Subsequently, signals relay to the nucleus, promoting the synthesis of IFN-Is, which then bind to IFNARs, activate the JAK/STAT pathway, leading to inhibition of mRNA translation, and RNA degradation. Adaptive immunity: antigen-presenting cells process the replicon vaccine particles to expose the recombinant subgenome and produce antigens. Antigen fragments presented in MHC-I and MHC-II activate cytotoxic T lymphocytes (CTLs) and helper T cells respectively. By combining the signals that originate from B cell receptors with the interaction of the integral antigen and the signals from helper T cells, B cells are activated

Innate immune response

Upon administration, repRNAs infect cells and localize in the cytosol or endosomes, binding to different pattern recognition receptors, and activating different innate sensing pathways. In the cytosol, short dsRNA containing a 5’ triphosphate end interacts with RIG-I [107], and long dsRNA interacts with MDA-5 [108]. Stimulation of these two receptors prepares the caspase activation and recruitment domain (CARD) of the receptor for binding the mitochondrial antiviral-signaling protein (MAVS), eliciting downstream reactions and synthesis of type I IFNs and pro-inflammatory cytokines. Endosomal dsRNA interacts with TLR-3, and uridine-rich ssRNA interacts with TLR7/8 [109]. Adaptor proteins, TIR-domain-containing adaptor-inducing IFN-β (TRIF) and myeloid differentiation primary response gene 88 (MyD88), relay the signals from the receptors to the downstream pathways, involving mediators like tumor necrosis factor receptor-associated factor (TRAF) proteins and the IkB kinase (IKK) complex [110]. These mediators subsequently trigger the nuclear transport of transcription factors IRF3, IRF7 and NF-κB, which leads to production of type I IFNs, pro-inflammatory cytokines, and expressions of a subset of interferon-stimulated genes (ISGs) [110]. The release of type I IFN activates interferon-α/β receptors (IFNARs), causing activation of the JAK/STAT pathway, prompting the expression of interferon-stimulated genes, including dsRNA-dependent protein kinase (PKR), and 20–50-oligoadenylate synthetase (OAS), which undermines mRNA translation and promotes mRNA degradation [110]. Thus, the innate immune response-stimulating property of repRNAs may support or hamper therapeutic outcomes as adjuvants or promotors of mRNA degradation, respectively. In addition, immune responses triggered by transfection of repRNA can induce adverse effects ranging from flu-like symptoms to the potential risks of autoimmune diseases. Therefore, different factors and conditions should be considered to optimize the eventual outcome of repRNA vaccination [110].

On one hand, studies have demonstrated that inhibiting innate immunity can enhance the expression of repRNA-delivered GOI. Ruxolitinib, a potent inhibitor of JAK, enhances the in vivo protein expression of repRNA [111]. Awe et al. demonstrated that BAY11, an inhibitor of the IKK complex, was more effective than BX795 (a TBK1 and IKKε inhibitor) in enhancing protein expression and stabilizing modified mRNA, primarily through the downregulation of NF-κB expression [112]. Dexamethasone enhanced luciferase expression following the intravenous delivery of dexamethasone-palmitate-loaded mRNA lipoplexes, likely due to the NF-κB inhibitory activity [113]. Zhong et al. investigated another corticosteroid, clobetasol propionate, which effectively reduced type I interferon responses and significantly boosted expression when used in intradermal electroporation of a repRNA vaccine targeting the ZIKV [114]. Introducing innate inhibiting proteins like PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a into repRNA encoding GOI was tested effective in elevating GOI expression 100- to 500-fold in vitro. The MERS-CoV ORF4a protein partially mitigates the dose nonlinearity observed in vivo [111]. On the other hand, Manara et al. stated that increasing recruitment of antigen-presenting cells by co-administration of repRNA encoding GM-CSF and repRNA encoding GOI led to improved GOI-specific CD8⁺ responses, which enhanced vaccine efficacy [115]. Furthermore, to evade innate immune responses triggered by repRNA vaccination and elevate the GOI expressions, compatible modified nucleoside triphosphates, including hydroxymethylcytidine (hm5C), 5-methylcytidine (m5C) or 5-methyluridine (m5U), are designed, 100% substitution of which in repRNA significantly suppresses the expression of IFN-related genes (IFNα1, IFNα2 and IFNβ1) and preserves the translation activities in infected cells. 10 ng dose of m5C repRNA vaccine encoding spike (S) protein of SARS-CoV-2 conferred significant protection against lethal viral challenge compared to the wild-type (WT) repRNA vaccinated animals [116].

Adaptive immune responses

The antigen, which is overexpressed as a result of repRNA immunization, is internalized by antigen-presenting cells and subsequently presented on the major histocompatibility complex (MHC) molecules, encompassing both MHC class I and MHC class II [117]. The interaction of these with the T cell receptor activates T cells. The signal conveyed by MHC-II activates CD4⁺ T cells, and the signal conveyed by MHC-I activates CD8⁺ T cells [117]. Alwis et al. immunized mice with a single dose of VEEV-based replicon vaccine against SARS-CoV-2, and detected strong cellular immunity responses, including increasing antigen-specific CD8⁺ T cells, and IFN-γ and IL-4-positive CD4⁺ T helper lymphocytes [118]. Moreover, repRNA vaccines are proven to enhance immune memory. Johnson et al. have demonstrated VSV-vectored respiratory syncytial virus (RSV) enhanced the percentage of effector memory cells in the T cell population, which deploy immediate protection after re-infection [119]. Furthermore, Rattanasena et al. implied that immunization with a heterologous KUNV replicon VLP/vaccinia virus (VACV) vaccine induced higher central memory responses than a heterologous DNA/VACV vaccine immunization strategy, which confers long-term protection [120].

Pre-clinical studies: RepRNA vaccines against human infectious diseases

Coronavirus replicon vaccines

Severe acute respiratory syndrome coronavirus 2

In 2019, a novel coronavirus caused a large number of viral pneumonia cases in Wuhan, China. On February 11, 2020, the International Committee on Taxonomy of Viruses named this new coronavirus “SARS-CoV-2”, and the World Health Organization named the disease it causes “COVID-19.” According to the COVID-19 dashboard by the center for systems science and engineering at Johns Hopkins University, as of August 11, 2020, more than 20 million COVID-19 cases had been reported in 216 countries and regions across six continents, with over 733,000 patient deaths [121]. In addition to viral pneumonia, SARS-CoV-2 infection can also lead to multi-organ dysfunction, including the respiratory tract, heart, gastrointestinal tract, kidneys, and brain. [122]. In response to the pandemic, mRNA vaccines developed from Pfizer/BioNTech (BNT162b2) and Moderna (mRNA-1273) were granted emergency use authorization by the FDA in 2020. Both vaccines encode the SARS-CoV-2 S glycoprotein and are delivered by LNP [123]. In 2020, Erasmus et al. developed a SARS-CoV-2 vaccine using alphavirus-derived repRNA with the attenuated VEEV TC-83 strain as a vector, incorporating the full-length SARS-CoV-2 S protein sequence. The repRNA was combined with a LION emulsion containing cationic squalene and superparamagnetic iron oxide nanoparticles. This intramuscular vaccine prompted strong production of anti-SARS-CoV-2 S protein IgG antibodies and the antibody responses lasted over 70 days with neutralizing titers similar to those in convalescent COVID-19 patients [104]. Due to the concern that infection of SARS-CoV-2 in animal hosts may cause further mutations and impact viral epidemiology, Langereis et al. tested the immunogenicity of VEEV-vectored SARS-CoV-2 vaccine expressing S protein between cats. The vaccine potentiated high titers of neutralizing antibodies and prevented virus presentation in oropharyngeal or nasal swabs upon SARS-CoV-2 challenge [124]. Mohammadi et al. utilized an alphavirus-based S protein-expressing vaccine formulated with LNP to be delivered orally. The immunization with the vaccine elicited humoral immunity against both the SARS-CoV-2 delta (B.1.617 lineage) and alpha (B.1.1. 7 lineage) variants in mice. [125]. Native S protein sequences from SARS Cov-2 A.1, B.1 variants and prefusion S protein sequences from A.1, B.1, B.1.1.7, B.1.351 were inserted into the VEEV backbone, respectively, and delivered by LION. A.1 spike-vaccinated mice showed equivalent neutralization activities against homologous A.1 challenge and heterologous B.1 challenge. Native- or pre-fusion B.1-spike-vaccinated mice demonstrated no significant difference in neutralization activities between A.1, B.1, and B.1.1.7. Both mice and hamsters showed that B.1.351 is the most vaccine-resistant strain among the tested. Moreover, vaccinated hamsters showed significantly lower levels of infectious virus shedding when exposed to heterologous SARS-CoV-2 variants [126]. O’Connor et al. designed an immunodeficient mice model. The immunization of a single dose of COVID-19 repRNA vaccine expressing S protein (A.1) from SARS-CoV-2 (repRNA-CoV2S) can elicit moderate T cell and antibody responses, suggesting the applicability of the vaccine in HIV-infected populations [127]. Furthermore, using non-human primates, O’Connor verified that under the circumstances where neutralizing antibody responses have waned to undetectable levels, repRNA vaccine encoding S protein still provided durable protection against SARS-CoV-2 and decreased the viral shedding [128]. Based on the backbone genome of the SINV alphavirus vector, Scaglione et al. designed an S protein-expressing vaccine against SARS-CoV-2, which when co-injected with an OX40 immunostimulatory antibody, elicited sustained neutralizing antibodies and strong T cell responses in mice [129]. Amano et al. introduced a five amino acid insertion (TGAAA) between amino acids 586 (N) and 587 (T) of the VEEV nsP2 protein. This mutated VEEV vector was screened and tested to keep GOI expressed at 30–35 °C but inactive at ≥ 37 °C under the subgenomic promoter. This temperature sensitivity makes the repRNA vaccine ideal for intradermal use when it can engage more antigen-presenting cells and elicit stronger T-cell responses compared to intramuscular or subcutaneous methods. Temperature sensitivity helps avoid systemic toxicities. Although the first immunization does not trigger a humoral immune response, antibodies can be produced with further antigen stimulation or a booster vaccination [130]. Besides, Li et al. demonstrated that incorporating SARS-CoV-2 neutralizing antibody sequences into the VEEV genome can create a vaccine against respiratory infection of SARS-CoV-2 via intranasal delivery. This VRP-based vaccine sustains CB6 antibody expression in the lungs for over 5 days and targets most major lung cell types in mice [131]. To enhance the delivery efficacy of the repRNA vaccine, researchers constructed new delivery platforms including the Salmonella bactofection approach [106], coatsome-replicon vehicles [101], and self-assembling liposome-protamine-RNA nanoparticles [132], which were all reported safe and efficient (Fig. 6).

Fig. 6

The grouped infectious diseases that have been studied in replicon vaccine studies. The structures of microorganisms are illustrated on the left side, while the information from preclinical studies is summarized on the right side. The blue frame denotes the GOI that were incorporated into replicon vaccines, the yellow frame indicates the viral vector employed, and the red frame delineates the outcomes following immunization

In addition to alphavirus vectors, other viral vectors have also been utilized to manufacture repRNA vaccines against SARS-CoV-2. Sun et al. developed a live virus vaccine candidate for SARS-CoV-2 using NDV as the viral vector, incorporating wild-type and membrane-anchored S protein sequences. This NDV-based vaccine produced strong neutralizing antibodies and protected mice from SARS-CoV-2 without detectable virus in the lungs. It also offers cost-effective production in chicken eggs compared to alphavirus vectors. Additionally, NDV is not a human pathogen, thus pre-existing immunity in humans does not interfere with the delivery of GOI [82]. Sun reported a modified NDV vector containing the prefusion-stabilizing Hexa Pro mutations in the S construct resulted in optimized expression in mammalian host cells and enhanced immunogenicity of the vaccine [83]. Hennrich et al. developed a SARS-CoV-2 vaccine using a VSV vector that expresses a chimeric minispike combining the S protein’s receptor-binding domain (RBD) with a rabies virus-derived transmembrane sequence. This design enables cell surface RBD expression and non-infectious VLPs, leading to high neutralizing antibody levels in mice after one dose, with further increases after a booster immunization [133]. Oreshkova et al. designed vaccine candidates by substituting prM and E protein sequences of the YFV genome with the S1 or S2 domain of the S proteins from MERS-CoV, SARS-CoV, or mouse hepatitis virus, respectively, among which candidates expressing the S1 domain of MERS-CoV and SARS-CoV elicited humoral immune responses against the antigen and the NS1 protein of the YFV replicon [134]. The application of YFV replicon is verified by Nakamura et al., who inserted the ectodomain S protein sequence between the prM and E sequence of the replicon genome. The vaccine evokes significant T-cell responses (Fig. 6) [63].

Middle East respiratory syndrome coronavirus

Infection induces variable clinical manifestations from asymptomatic to severe pneumonia that accompanies acute respiratory distress, septic shock, and renal failure [135]. Gutierrez-Alvarez et al. introduced an attenuated modified MERS-CoV replicon, of which the E gene and the accessory ORF3, 4a, 4b, and 5 were deleted. After the modification, the replicon is replication-competent but propagation-defective. Immunization with this attenuated replicon induced sterilizing immunity against lethal MERS-CoV challenges, with no pathological changes observed in the lung histology. Additionally, the vaccination evoked robust neutralizing antibody responses (Fig. 6) [136].

Ebola and Lassa virus replicon vaccines

Lassa virus (LASV) and EBOV from the Arenaviridae and Filoviridae families, respectively, cause acute and fatal hemorrhagic fever. Using the VEEV replicon backbone, Pushko et al. designed LASV monovalent vaccines expressing either LASV nucleoprotein or glycoprotein, and bivalent vaccines against EBOV and LASV in two forms: the combination of LASV glycoprotein (LGP)- and EBOV glycoprotein (EGP)-VRP, or the dual-expression EGP/LGP-VRP. The LASV monovalent replicon vaccine protected guinea pigs from lethal infection by LASV. Furthermore, both bivalent vaccines offered protection against both EBOV and LASV [137]. Reynard et al. reported that the KUNV-based replicon vaccine expressing WT glycoprotein (GP) or mutated GP (D637L) of the EBOV provided dose-dependent protection against lethal challenge with EBOV in guinea pigs [138]. In nonhuman primates, the KUNV-based replicon vaccine expressing the mutated EGP (D637L) was later proven to protect the majority of immunized animals from the lethal EBOV challenge [139]. The viral-cell fusion of the EBOV is mediated by the gH/gL glycoprotein complex, the full length of which has proven to be an effective antigen candidate to be expressed in the alphavirus replicon-based vaccine. The vaccination of this designed RNA sequence elicited high neutralizing antibody titers and robust vaccine-specific CD8⁺ T cell responses, protecting humanized mice from lethal EBOV challenge [94]. By replacing the S segment coded glycoprotein precursor (GPC) gene of the LASV with the gene for the fluorescent protein ZsG, Kainulainen et al. produce LASV VRPs that only causes a single round of infection. Vaccination with VRPs prevented clinical manifestations of lethal LASV in guinea pigs [140]. Kainulainen et al. later deleted the fluorescent protein ZsG and left 9 bases between the untranslated region and the intergenic region, which encodes two stop codons, to further enhance the VRP yields. The refined VRP illustrated complete protection from LASV infection (Fig. 6) [141].

Simian/human immunodeficiency virus (SIV/HIV) replicon vaccine

HIV infection leads to the depletion of CD4⁺ T cells, macrophages, and monocytes in humans, destroying the immune system, and exposing patients to the hazards of fatal infectious diseases. SIV causes persistent infections in nonhuman primates and resembles HIV [142]. TAB9 is a multi-epitope polypeptide comprise the fusion of the central region of the gp120V3 loop from HIV-1 isolates LR150, JY1, RF, MN, BRVA, and IIIB, and the sequence encoding the N-terminal part of the p64K protein from Neisseria meningitidis. Prime vaccination with an SFV-based vaccine expressing the TAB9 multi-epitope followed by a booster immunization with a recombinant fowlpox vaccine expressing TAB9 significantly reduced VVTAB13 replication, an HIV-1 recombinant vaccinia virus expressing TAB13 protein [143]. The TAB13 sequence comprises the TAB9 sequence with the addition of two extra V3 loops inserted between the BRVA and IIIB epitopes [143]. Chimeric alphavirus replicon particles have been engineered with both VEEV and SINV to generate replicon particles with optimal potency and safety. Xu et al. applied this chimeric viral vector to express SIVgag (p55), HIVEnv (gp140). The co-administration of SIVgag and HIVEnv replicon vaccine evoked anti-SIVgag and anti-HIVEnv-specific IFN-γ responses and high titers of binding and neutralizing antibodies against HIVEnv in non-human primates, but not SIVgag. A booster vaccination with recombinant trimeric HIVΔV2gp140Env protein further enhanced the HIV Env-specific IFN-γ responses and the neutralizing antibody titers. Animals immunized with this prime-boost regimen displayed significantly lower primary plasma viremia levels than the control animals [144]. Considering the induction of effector memory and central memory responses, mediation of protection, and insert stability, Anraku et al. tested four different KUNV replicon SIVmac239 gag vaccines expressing a WT gag gene, an RNA-optimized gag gene, a codon-optimized gag gene and a modified gag-pol gene construct respectively. The gag-pol expressing candidate performed best [58], which was later utilized in the vaccination of macaques. The results suggested that the immunization elicited KUNV-specific antibody responses with no thread of SIV-specific T-cell immunity [145]. Using the LION-delivered ZIKV replicon vaccine expressing the Env protein of HIV-1, Khandhar et al. proved that the antibody responses induced in immunized maternal rabbits were able to be transferred to the offspring (Fig. 6) [146].

Flavivirus replicon vaccines

Zika virus

Zika virus is a member of the Flaviviridae family. The infection of ZIKV can cause neurological complications and memory and cognitive deficits in adults. The vertical transmission of ZIKV endangers pregnant women with high-risk miscarriages and fetuses with microcephaly or other brain abnormalities [95]. Additionally, the manufacturing of flavivirus vaccines has encountered obstacles resulting from the Ab-dependent enhancement (ADE), which is that suboptimal antibody responses against a type of flavivirus enhance the severity of the subsequent infection of a heterologous flavivirus infection. The mechanism behind ADE involves the sub-neutralizing antibodies supporting the uptake of the virus into the Fcγ receptor-bearing cells [147,148,149]. Upon ZIKV infection, T-cell immune responses are mainly generated against ZIKV nsP3, while the antibody immune responses are mainly triggered by the ZIKV prM-E protein. Using the Japanese encephalitis virus (JEV) replicon, Yamanaka et al. successfully generated single-round infection particles expressing prM and E of ZIKV [150]. Based on the VEEV backbone, Ngono et al. designed a repRNA vaccine candidate encoding the nsP3 protein of ZIKV, which employed polyfunctional CD8⁺ T cells but no neutralizing antibody response, bypassing the promoting factors of ADE [95]. The vigorous CD8⁺ T cells successfully prevented death and vertical transmission in mice upon lethal ZIKV challenge [95]. Erasmus et al. developed another VEEV-based replicon candidate expressing a highly neutralizing human mAb, ZIKV-117, which elicited high levels of antibody titers in mice that resulted in protection against lethal ZIKV challenge (Fig. 6) [151].

Japanese encephalitis virus

JEV, is a flavivirus that leads to encephalitis in infected individuals in eastern and southern Asia. Pigs are intermediate hosts in the zoonotic transmission cycle between birds and mosquitoes of JEV [152, 153]. Therefore, the restraint on the transmission of porcine can prevent JEV epidemics in humans. Therefore, Yang et al. manufactured a classic swine fever virus (CSFV)-based VRP vaccine expressing a truncated envelope protein (amino acid 292–402, domain III) of JEV [154], which has been demonstrated to elicit neutralizing antibodies and protective immunity [155, 156]. The vaccination with VRP generated robust production of JEV-specific antibodies and conferred protection against lethal JEV infection in both mice and swine models [154]. By deleting the structural protein region of the JEV subgenomic replicon, a JEV replicon vaccine vector was developed by Huang et al. This viral vector cooperated with an engineered BHK packaging cell line, which stably expresses C, prM, and E proteins of JEV, led to encapsidated JEV propagation-deficient pseudoinfectious particles. The 3-round vaccinations of pseudoinfectious particles boosted the JEV-specific and neutralizing antibody titers. Using the antisera of immunized mice to vaccinate susceptible weanling mice, a 75% survival rate was reached against the lethal JEV challenge (Fig. 6) [157].

Dengue virus

The infection of DENV (4 serotypes: DENV1-4), a type of flavivirus, causes fever, headache, muscle and joint pains, rash, nausea, and vomiting. Severe infections induce dengue hemorrhagic fever (DHF) that might jeopardize the lives of patients by promoting vascular permeability and the occurrence of dengue shock syndrome (DSS), which once happened, the fatality rate raised to 44% [158]. Infection with one serotype of DENV induced production of both type-specific antibodies and cross-reactive antibodies. The cross-reactive antibodies may protect against or exacerbate subsequent symptomatic or severe DENV infections following both natural infections and vaccination, underscoring the comprehensive evaluation of DENV vaccines [159]. Targeting broad protective immunity, tetravalent DENV vaccines, including Dengvaxia, TAK-003, and Butantan-DV, have shown positive results, but failed to generate equivalent protection against the four serotypes [160]. Chen et al. engineered a replicon vaccine candidate encoding DENV1 prM and E proteins based on the VEEV VRP system. Using two doses of a conventional plasmid DNA vaccine expressing the same antigen as prime vaccinations and one dose of the VEEV VRP vaccine as booster vaccination, higher DENV type-1 specific IgG responses and neutralizing antibody titers were elicited compared with homologous three-dose plasmid DNA/VEEV VRP vaccination. Despite all three vaccination regimens–-homologous three-dose plasmid DNA vaccination, homologous three-dose VEEV VRP vaccination, and the heterologous prime-boost regimen, significantly protected the mice from viremia, only the heterologous regimen displayed complete protection. Low levels of cross-neutralizing antibodies were detected from all three vaccination groups on day 140, and diminished to undetectable levels by day 252 [161]. Furthermore, using the VEEV VRP system, White et al. designed a DENV vaccine expressing prM and E from a mouse-adapted DENV2 strain. The first dose vaccination of this VEEV VRP vaccine induced the production of high titers of DENV2-specific IgG and neutralizing antibodies in 3-week-old weanling BALB/c mice. The second dose further enhanced the neutralizing titers, which have long-term preservation for at least 30 weeks [162]. Studies have suggested that the subneutralizing levels of maternal anti-DENV antibodies are associated with the occurrence of DHF/DSS in the first year of life of newborns [163,164,165]. The VEEV VRP-based DENV vaccine designed by White et al. has shown the capability to evoke neutralizing antibody responses regardless of the maternal immune status in mice (Fig. 6) [162]. The repRNA DENV vaccine candidates lack broad protection against DENV1-4, hindering further use in human populations. However, the attempts to apply repRNA in constructing DENV vaccines provide a theoretical basis for the development of a repRNA-based tetravalent dengue vaccine.

Murray Valley encephalitis virus

Murray Valley encephalitis virus (MVEV) is a flavivirus that causes infrequent outbreaks of encephalitis around the Murray Valley region of south-eastern Australia and is usually associated with high mortality [166]. Colombage et al. utilized SFV replicon backbone to design MVEV prM and E gene-expressing DNA replicon vaccines. Long-lasting neutralizing antibody responses and protection against MVEV exposure were observed through gene-gun-mediated subcutaneous delivery. Intramuscular injection of the vaccine candidate elicited the production of neutralizing antibodies but did not restrain the viral infectivity (Fig. 6) [167].

Hepatitis C virus

Hepatitis C virus (HCV) damages liver tissues, arousing global public concerns due to it being a risk factor for cirrhosis and hepatocellular carcinoma [168]. María et al. generated an alphavirus-based DNA replicon vaccine encoding either structural Core-E1-E2 or nonstructural p7-nsP2-nsP3 HCV proteins, which was later applied as the prime dose with subsequent recombinant modified vaccinia virus Ankara (MVA) vector expressing the nearly full-length genome of HCV as a booster dose. This heterologous regimen elicited long-lasting HCV-specific CD4⁺ and CD8⁺ T cell immune responses that are polyfunctional, with the effector memory phenotypes targeted E1-E2 and nsP2-nsP3 respectively. Besides, this heterologous regimen outperformed the homologous MVA-HCV immunization in cellular and humoral immune responses (Fig. 6) [169].

Paramyxovirus replicon vaccines

Human metapneumovirus

The infection of human metapneumovirus (hMPV), which belongs to the Paramyxoviride family, leads to upper and lower respiratory tract infections, which range from mild upper respiratory tract disease to severe bronchiolitis and pneumonia, targeting infants, the elderly, and immunocompromised individuals. Mok et al. designed VRP vaccine candidates expressing hMPV F or attachment glycoproteins based on the VEEV replicon backbone [170]. The result showed that only the vaccine encoding hMPV F protein induced the production of F-specific neutralizing antibodies and the secretion of IgA at the respiratory mucosa, which promoted the reduction of viral replication in the respiratory tract upon hMPV challenge (Fig. 6) [170].

Measles

In developing countries, measles continues to increase health burdens by endangering the lives of infants and young children [171]. Attenuated MV vaccines exhibit low potency during the ‘‘window of vulnerability,’’ between four and nine months of age, when the maternal inherited immune protection against MV is impaired, and the immune system of infants has not yet been established [172]. Capozzo et al. developed DNA replicon vaccines using a SINV backbone, encoding MV H (pMSIN-H) or both H and F proteins (pMSINH-FdU). Neonatal immunization with the pMSIN-H vaccine achieved long-term, high avidity neutralizing antibodies at adult-like levels and prevented syncytium formation, even with placentally-transferred antibodies present. The study indicated that these vaccines successfully triggered Th1-type immune responses without interference from maternal antibodies [173]. Pasetti et al. utilized these SINV-based measles vaccines for prime immunization followed by a booster vaccination with a licensed measles vaccine and successfully observed protection against clinical measles and viremia after WT virus challenge in infant or juvenile rhesus macaques [174]. Pan et al. developed a chimeric VEEV/SINV replicon system, for which vaccine candidates express H or H and F proteins. The vaccination with both vaccine candidates induced high titers of MV-specific neutralizing antibodies and IFN-γ-producing T cells. Moreover, they both provided complete protection from viremia and rash in juvenile and infant monkeys after the MV challenge (Fig. 6) [175].

Parainfluenza virus 3

Human parainfluenza virus type 3 (PIV3) causes diseases in immunocompromised individuals. It leads to bronchiolitis and pneumonia in infants or young children, and severe lower respiratory tract illness in adults. A VEEV/SINV chimeric alphavirus vector expressing the PIV3 hemagglutinin-neuraminidase glycoprotein displayed high immunogenicity in mice and hamsters, evoking robust neutralizing antibody responses against PIV3 [176]. Administration through the intramuscular or intranasal route prevented virus replication in both the upper and lower respiratory tract in hamsters (Fig. 6) [176].

Respiratory syncytial virus

RSV causes severe lower respiratory tract disease in a seasonal pattern, attacking all age groups, but burdens infants, the elderly, and individuals with immunosuppression or chronic cardiopulmonary the most. Elliott et al. developed VEEV replicon-based vaccine candidates targeting RSV’s Fa or attachment a or attachment b proteins from subgroups A and B. Intramuscular administration of these candidate vaccines in mice and rhesus macaques resulted in high serum neutralizing antibody levels against RSV strains A and B [177]. In mice, the vaccine also promoted balanced Th1/Th2 responses, shown by increased RSV-specific IFN-γ⁺ splenocytes, reduced lung eosinophils and type 2 T cell cytokines, and enhanced lysis of RSV protein-loaded cells. The cocktail of replicon RSV vaccines protected mice from the intranasal challenge of RSV [177]. Similar results were obtained for VEEV VRP-based vaccines encoding RSV F or attachment glycoproteins. During the intranasal challenge, the F protein expressing VRP vaccination reduced viral titers in both the upper and lower respiratory tracts below the detection level, while the attachment protein VRP-vaccinated animals had viral replication in the upper but not lower respiratory tract (Fig. 6) [178].

Bunyavirus replicon vaccines

Rift Valley fever virus

Rift Valley fever virus (RVFV) is a mosquito-transmitted bunyavirus. Human infections are mostly benign, while a small proportion (0.5–1%) suffers from fatal encephalitis or hemorrhagic fever. RVFV has a single-stranded RNA consisting of three segments: the large, medium, and small segments. The medium segment encodes a non-structural protein m (NSm) and a polyprotein precursor of the Gn and Gc glycoproteins [179]. Gn and Gc glycoproteins are responsible for the formation of envelope peplomers, which are vital in viral binding and entry. Heise et al. designed a SINV vector repRNA vaccine, with the medium segment sequence incorporated downstream of the 26S promotor. Vigorous anti-RVFV antibody responses were detected in both immunized mice and sheep. After a prime-boost vaccination regimen, 100% protection against RSFV challenge was obtained (Fig. 6) [179].

Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus that causes severe hemorrhagic fever. Based on the VEEV backbone, Leventhal et al. designed repRNA vaccines expressing either the CCHFV nucleoprotein (repNP) or glycoprotein precursor (repGPC) [180]. Results demonstrated that repNP vaccination provided protection upon CCHFV challenge, with little clinical manifestation. The combination of repNP and repGPC protected against clinical disease and viral replication. RepNP vaccination evokes mainly antibody responses while repGPC immunization evokes mainly cellular immune responses (Fig. 6) [180].

Bacterial replicon vaccines

Anthrax, botulism

Clostridium botulinum produces highly toxic neurotoxins of different serotypes, from serotypes A to G, among which serotype A is the most toxic and is associated with human botulism. Each toxin has a structure that includes a heavy chain (Hc) and a light chain joined by a single disulfide bond. The carboxyterminal of the Hc participates in the binding to neurons and is not toxic, indicating a possible immune eliciting effect [181]. Using a DNA intermediate, Yu et al. developed a Clostridium botulinum replicon vaccine encoding the Hc domain of neurotoxin serotype A (AHc) [181]. The recombinant DNA replicon induced apoptosis of transfected cells. Furthermore, the replicon-vaccinated mice significantly outperformed the conventional plasmid-vaccinated mice in the antibody responses, cellular immune responses, and protection against botulinum neurotoxin serotype A (BoNT/A) intoxication, with the same antigen being expressed [181]. Because of the threat of bioterrorism, Lee et al. designed three VEEV-based VRP vaccines against anthrax, Marburg fever, or botulism, encoding mature 83-kDa protective antigen from Bacillus anthracis, the GP from Marburg virus (MBGV), or the H_C fragment from botulinum neurotoxin (BoNT H_C), respectively. Vaccination with monovalent VRP vaccine or with trivalent replicon vaccine formulated with the three types of VRPs both aroused antibody responses that target the corresponding replicon-expressed antigen. A mixture of replicon vaccines expressing BoNT H_C serotype C (BoNT/C H_C) and mature 83-kDa protective antigen offered 80% and 100% protection against B. anthracis (Sterne strain) and BoNT/C challenges in mice, respectively (Fig. 6) [182].

Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiological agent of tuberculosis, causes numerous deaths in tropical regions [183]. Dalmia et al. constructed a DNA-based VEEV replicon vaccine that expressed a fusion of the mycobacterial antigens α-crystallin and antigen 85B, the administration of which potently inhibited bacterial growth in both lungs and spleens. This vaccine elicited both CD4⁺ and CD8⁺ T cell responses for at least 10 weeks after immunization, which was suggested crucial in effective immunity against Mycobacterium tuberculosis (Fig. 6) [184].

Mycobacterium avium

Mycobacterium avium complex (MAC) infections make up 85–90% of all nontuberculous mycobacterial (NTM) pulmonary disease cases [185]. Chronic disease caused by NTM increases the length of treatment, which can elevate treatment-associated toxicity, incomplete treatment, or recurrence of disease [186]. Using a repRNA/ LION delivery system, Rais et al. constructed a MAC vaccine candidate expressing ID91, which consisted of four bacterial antigens: Rv3619, Rv2389, Rv3478, and Rv1886, or ID91 + GLA-SE. GLA-SE, a glucopyranosyl lipid adjuvant formulated in an oil-in-water stable nano-emulsion, is a synthetic TLR4 agonist adjuvant. Prime vaccination with ID91/ ID91 + GLA-SE replicons and booster vaccination with the ID91 + GLA-SE protein vaccine elicited stronger immune responses against MAC compared to homologous vaccinations. This strategy also provided higher polyfunctional CD4 + TH1 immune response and greater pulmonary protection. Besides, the heterologous regimen aroused IFN-γ and TNF-secreting CD8⁺ T cells, high systemic proinflammatory cytokine responses, and Ag85B-specific humoral antibody responses (Fig. 6) [187].

Lyme disease

Lyme disease is transmitted by Borrelia burgdorferi through the bite of infected Ixodes ticks. The early stage of Lyme disease is localized and characterized by erythema migrans or homogenously erythematous lesions. The early disseminated phase involves the occurrence of secondary EM, acute carditis, nervous system symptoms (e.g., radiculopathy, meningitis, facial nerve palsy, and other cranial neuropathies), and brief attacks of monoarticular or oligoarticular arthritis. The late disseminated phase sees more advanced joints and nervous system damage, including monoarticular and oligoarticular arthritis, peripheral neuropathy, or encephalomyelitis [188]. Gipson et al. designed a VEEV VRP-based replicon vaccine expressing Borrelia burgdorferi outer surface protein A (OspA), vaccination of which led to significant production of anti-OspA antibodies, and Th1 type immune responses (Fig. 6) [189].

Other infectious diseases studied in replicon vaccine studies

Chikungunya virus

CHIKV causes chikungunya fever, which is characterized by long-term debilitating arthralgia that progresses to chronic arthritis [28]. Taylor et al. developed a repRNA vaccine by replacing 10 WT amino acids with alanines of the nucleolar localization sequence (NoLS) of CHIKV capsid protein, attenuating the viral replication [190]. One dose of liposome-delivered CHIKV-NoLS RNA prevented severe CHIKV-induced footpad swelling from the CHIKV-WT challenge in the ipsilateral foot, providing local protection [28]. However, compared to the NoLS mutated live-attenuated vaccine, the repRNA vaccine showed less enhancement of antibody responses, which is suspected to result from the liposome adjuvant [28]. Similarly, Szurgot et al. deleted 183 nucleotides in the nsP3 gene, which reduces the viral replication and attenuates the viremia after CHIKV infection, to manufacture a vaccine candidate. The vaccination evokes significant antibody responses, with high titers in both binding and neutralizing antibodies which protect mice from viremia after the CHIKV challenge (Fig. 7) [191].

Fig. 7

The individual infectious diseases that have been studied in replicon vaccine studies. The left side of the figure shows the structures of microorganisms, while the right side illustrates information from preclinical studies, with the GOI incorporated into replicon vaccines demonstrated in the blue frame, the employed viral vector narrated in the yellow frame, and the outcomes following immunization summarized in the red frame

Malaria

Malaria is a devastating infection caused by Plasmodium parasites, the symptoms of which are non-specific including fever, chills, headache, myalgia, nausea, vomiting, diarrhea, fatigue, abdominal pain, and altered mentation. Severe cases cause serious vital organ damage, leading to shock, pulmonary edema, massive bleeding, seizures, impaired consciousness, etc. [192]. MacMillen et al. constructed a repRNA vaccine utilizing the VEEV vector to express full-length CS of Plasmodium yoelii, which was formulated with the LION nanoparticle carrier. The synthetic replicon vaccine was intramuscularly vaccinated as a prime dose, and a radiation-attenuated PE sporozoite (RAS) whole-organism (WO) vaccine was intravascularly injected as the trapping dose on the same day or with a five-day interval. This vaccination regimen induced both cellular and humoral immune responses. The same-day immunization regimen conferred complete protection in 90% of vaccinated animals against a sporozoite challenge performed 3 weeks later (Fig. 7) [193].

Norovirus

Norovirus, a highly infectious virus, is the leading cause of non-bacterial gastroenteritis outbreaks. While it is self-limiting in those with healthy immune systems, it can cause severe complications in immunocompromised individuals. Its rapidly evolving genome and antigens complicate vaccine development [194]. Therefore, the effective norovirus vaccine should elicit immune responses against various norovirus strains [195]. An alphavirus-based multivalent norovirus VLP encoding norovirus ORF2 was developed by Lobue et al. Vaccination of the multivalent vaccine-induced antibody responses to heterologous human stains outside of the vaccine component and reduced viral loads upon viral exposure. The involvement of alphavirus enhanced systemic and mucosal immunity compared to low-dose CpG DNA (Fig. 7) [195].

Smallpox

Current vaccines for smallpox are not appliable for a large population, leading to demands for a new, safe, and effective vaccine [196]. Hooper et al. developed a VRP expressing VACV A33R, B5R, A27L, and L1R genes based on the VEEV backbone [197]. The immunogenic effects were comparable to the live VACV. Immunized non-human primates developed strong antibody responses that neutralized and suppressed the spread of both the VACV and monkeypox virus. The administration of the vaccine also prevented severe disease caused by monkeypox (Fig. 7) [197].

Influenza virus

Influenza is a seasonal acute viral respiratory infection that normally causes outbreaks in winter [198]. Demoulins et al. designed influenza virus H5N1 nucleoprotein (Rep-NP) or hemagglutinin (Rep-HA) expressing influenza vaccines based on a non-cytopathogenic CSFV. The CSFV viral vector was deliberately chosen to target monocytes and DCs to prolong antigen expression in DCs. Both VRP and PEI–based formulation (polyplex) delivery systems elicited robust humoral and cellular responses against encoded antigens (Fig. 7) [100].

Rabies virus

Rabies virus is a zoonotic virus that can cause fatal encephalitic disease in humans [199]. A genetically modified SINV RNA replicon that expresses the rabies virus glycoprotein gene was constructed by Saxena et al. This repRNA vaccine performed similarly to the rabies bicistronic DNA vaccine encoding rabies virus glycoprotein [200] in the ability to elicit humoral and cellular responses and protection against challenges with the rabies virus CVS strain (Fig. 7).

Hepatitis B virus

Chronic hepatitis virus infection promotes the development of cirrhosis and hepatocellular carcinoma [201]. Singh et al., inserted hepatitis B virus (HBV) ORFs encoding HBV surface antigen (HBsAg) or dual protein HBsAg/HBV core antigen (HBcAg) into the replicon genome of MV. The recombinant MV viruses were immunogenic in genetically modified mice and could stably express the inserted antigen. Immunization of this recombinant virus evoked humoral responses against both MV and HBV (Fig. 7) [80].

Enterovirus 71

Enterovirus 71 (EV71), which attacks the central nervous system and may cause severe encephalitis, is prevalent in Asian countries infecting mainly the young population [202]. Huang et al. designed a JEV repRNA vaccine candidate expressing the neutralizing epitope SP70, which is conserved among sub-genome groups of different EV71 strains [203]. Moreover, vaccination led to complete dual protection against JEV or EV71, also in the offspring of immunized mice (Fig. 7) [203].

Preclinical research on replicon vaccines against cancers

Replicon vaccines have demonstrated anti-tumor efficacy through various mechanisms, such as the expression of tumor-specific and tumor-associated antigens and the expression of immunomodulatory factors. The successful enhancement of in vivo expression of the GOI offers promising prospects for personalized cancer treatment. In 1994, Ansardi et al. introduced a poliovirus replicon-based, CEA-expressing vaccine that successfully induced CEA-specific antibodies after the third vaccination [204]. In 2000, Leitner et al. applied a chimeric plasmid DNA vector derived from the SINV and SFV replicons in tumor antigens. The DNA replicon vaccine reached the conventional DNA plasmid vaccine immunogenicity level with 100 to 1000-fold lower doses than conventional plasmid DNA (Fig. 8) [205].

Fig. 8

The cancer types that have been studied in replicon vaccine studies. The schematic illustration of various cancers is displayed on the left side, while the information synthesized from preclinical studies is presented on the right side. The green frame indicates the gene GOI incorporated into the replicon genome, the blue frame represents the viral vectors utilized, and the pink frame depicts the outcomes following immunization

Cervical cancer

In 2019, the global incidence of cervical cancer reached 565,540.89, ranking the fourth highest incidence of malignancy [206]. Cervical cancer is strongly correlated with human papillomavirus (HPV) infections [207]. Recombinant vaccines including Cervarix, Gardasil, and GARDASIL 9 are approved by the FDA [208].

Cheng et al. used SINV repRNA to express HPV-16 E7 and discovered that targeting the endosomal/lysosomal compartment elicited stronger immune responses against cervical cancer metastasis compared to cytosolic/nuclear or secretary location. This was linked to increased apoptosis in transfected cells and E7 antigen loading in DCs [209]. Cheng et al. also found that by linking herpes simplex virus type 1 VP22 to E7 antigen boosts intercellular transport of replicon particles, enhancing the repRNA vaccine’s effectiveness [210]. This significantly increases E7-specific CD8⁺ T cell precursors and results in stronger protection against E7-expressing tumors compared to WT E7 SINV replicon particles [210]. Years later, Chen et al. developed a recombinant antigen combining the calreticulin (CRT) and E7 genes in a SINV replicon. This mutant vaccine boosted CD8⁺ T cell precursors and antitumor effects directly infecting DCs and promoting apoptosis, while also inducing immunological memory [211]. Lin et al. created a chimeric antigen by linking the mycobacterium tuberculosis HSP70 to the E7 protein. They demonstrated that the heterologous prime-boost vaccination regimen with SINV VRP expressing E7/HSP70 as the prime and the VACV-based E7/HSP70 vaccine as the booster showed potent antitumor effects that outperformed the DNA prime-vaccina boost strategy [212]. Velders et al. designed a VEEV VRP vaccine encoding E7. Mice were vaccinated on the seventh day post-tumor implantation, when all mice had developed palpable tumors. 67% of mice showed complete tumor regression after vaccination, which was maintained for 60 days [213]. Cassetti et al. designed a mutant gene sequence by the fusion of HPV E6 and E7 genes and inactivated its oncogenic potential by creating mutations at key residues for oncogenic function. The amino acids at positions ⁶³C and ¹⁰⁶C in E6, and at positions ²⁴C, ²⁶E, and ⁹¹C in E7, were replaced with glycine. VEEV VRP vaccines expressing mutant and WT fusion proteins were developed. Mutant and WT VRPs induced comparable CTL responses to an immunodominant E7_49–57 epitope, and comparable antitumor responses in E6⁺E7⁺ tumor challenge models [214]. van de Wall designed an SFV-based DNA replicon vaccine encoding E6,7, which illustrated significantly more potent antitumor effects compared with traditional DNA vaccines that expressed the same antigen, which did not halt the tumor outgrowth. After immunization with the DNA replicon vaccine, 85% of mice became tumor-free with a 200-fold lower equimolar dose than vaccination with a conventional DNA vaccine [215]. In addition to the intramuscular immunization route, van de Wall created a tattoo delivery route, through which the immunization of SFV replicon particles expressing E6 and E7 antigens contributed to more significant immune responses with tenfold lower transgene expression in the site of administration and draining lymph nodes [216]. Walczak et al. expressed concerns about the heterologous prime-boost regimen using an E6-E7 fusion gene SFV vaccine and E7 protein virosome. While this approach resulted in more antigen-specific CTLs than a homologous regimen, the increased vector-induced immunity did not enhance antigen responsiveness, cytolytic activity, or antitumor responses due to a rise in regulatory T cells (Treg). The lack of involvement of central memory T cells after immunization with the heterologous regimen could also weaken the antitumor effect [217]. Therefore, multifaceted evaluations need to be conducted before determining the potency of the heterologous regimen using viral vectors. Additionally, Kanodia et al. demonstrated that priming with LIGHT, a ligand for the lymphotoxin-β receptor that promotes lymphoid-like tissues inside the tumor microenvironment and recruits naïve T cells, could facilitate the antitumor effects of the HPV16 VRP vaccine, and eradicate established large cervical tumors [218]. The KUNV replicon was also applied to construct an expressing vaccine. Herd et al. constructed KUNV vectored vaccines encoding the CTL epitope of HPV16 E7 in three forms, naked RNA, plasmid DNA, and RNA packaged into VLPs. The VLP-delivered repRNA vaccine outperformed the rest two vaccines, eliciting high RAH/D^b-specific IFN-γ producing T cell responses, high functional CTL responses, and long-term memory responses. The prophylactic experiment showed that 80% of mice immunized with VLPs remained tumor-free for up to 70 days, whereas all unimmunized mice developed tumors. Mice that were completely protected were re-challenged with tumor, and remained tumor-free, indicating that the vaccine-induced immune protection was long-lasting (Fig. 8) [60].

Breast cancer

Breast cancer is one of the most common malignancies worldwide, the early phase of which is potentially curable, while the metastatic phase is incurable with available treatments [219]. So far, a triple-negative breast cancer vaccine has been approved by the FDA to be used in human clinical trials [220].

HER2/neu is a proto-oncogene that is amplified in a considerable proportion of breast cancer patients [221]. A SINV replicon-based plasmid vaccine encoding the sequence of neu was intramuscularly administrated to mice, and triggered robust antibody responses against the murine breast cancer cell line A2L2, inhibiting the distant metastasis of A2L2 cells. Further investigation claimed that antigen-specific Th1-type immune responses were recruited after the vaccination, and the immunization also inhibited spontaneous breast cancer development in transgenic mice [222]. Nelson et al. used a VEEV replicon to encode the rat neu sequence, which was shown to induce a restricted magnitude of immune responses that promote immune tolerance. The aid of the replicon delivery system overcame this obstacle, eliciting Th1 anti-tumor immune responses and immunological memory, providing 50% protection from tumor challenge [223]. Eralp et al. stated that chemotherapy with immunomodulating doses of doxorubicin or paclitaxel potentiated the antitumor efficacy of the VEEV VRP-based neu-expressing vaccine [224]. The potency of the VEEV replicon vector in breast cancer vaccine development was further demonstrated by Wang et al., which claimed that the VRP-neu vaccine provided 100% protection against breast tumor formation and associated death (Fig. 8) [225].

Melanoma

In 2020, 325, 000 new melanoma cases and 57, 000 melanoma-associated death occurred [226]. Melanoma is usually accompanied by a change of appearance, ulceration, and itchy of pigmented moles. It is the most lethal skin cancer type, the treatment of which is halted by resistance [227]. mRNA-4157/V940 vaccine from Moderna has been shown to induce a significant reduction in the risk of melanoma recurrence and death in combination with pembrolizumab [228].

A SINV viral vector encoding the melanocyte differentiation antigen gp100 and IL-18 was demonstrated to T cell and IFN-γ-mediated antitumor effects against brain-implanted melanoma in mice [229]. Targeting the murine cell adhesion molecule MUC18 that is upregulated during the progression of melanoma, a SINV DNA replicon vaccine combined with blocking Abs showed prophylactic effects against the incidences of melanoma development and its lung metastasis [230]. Recombinant VEEV VRP encoding human tyrosine was developed by Goldberg et al., which was administered as a boost following the prime dose, a plasmid DNA encoding the same antigen. The VRP vaccine candidate when used alone generated robust antibody and T-cell responses and delayed tumor growth, the involvement of which in the heterologous prime-boost regimen significantly enhanced the immune responses compared with a single treatment with the plasmid DNA vaccine [231]. Moreover, a VRP vaccine designed by Avogadri et al. that encoded the tyrosinase-related protein-2 (TRP-2) triggered long-term antitumor immune responses, including the activation of both IgG and CD8⁺ T cell effector responses. The activation of Fcγ receptors is regarded as crucial as well [232]. Based on this finding, Avogadri et al. also discovered that by combining Immunomodulatory mAbs that counteract CTLA-4, an inhibitory immune receptor, or activate glucocorticoid-induced TNF family–related gene (GITR), a costimulatory molecule with VRP–TRP2, the complete tumor regression rate raised to 90% and 50% respectively [233]. SFV-based DNA-launched replicon vaccine expressing the fusion protein of survivin and hCGb-CTP37, and adjuvant molecular protein GM-CSF enhanced the humoral and cellular immune responses against survivin⁺ and hCGb⁺ murine melanoma, delaying the tumor growth [234]. On top of that, Yin et al. constructed another SFV-based DNA replicon vaccine encoding 1–4 domains of murine VEGF receptor and IL-12 to be co-administered with the survivin and hCGb-CTP37-expressing DNA replicon vaccine. The combined therapy that targeted both tumor cells and angiogenesis more effectively repressed the tumor growth and elevated the survival rate (Fig. 8) [235].

Prostate cancer

Is the second most common cancer in men that results in high mortality. Age, race, family history, and germline mutations are confirmed risk factors for prostate cancer [236]. Sipuleucel-T, an autologous active cellular immunotherapy, is the first anti-cancer vaccine approved by the FDA [237] and approved to prolong the overall survival in metastatic castration-resistant prostate cancer patients [238].

Durso et al. constructed a VEEV-based prostate cancer vaccine expressing prostate-specific membrane antigen (PSMA), which was able to induce strong T-cell and B-cell responses, including Th-1 cytokines, strong CTL activity, and IgG2a/IgG2b antibodies, in a single dose of 2 × 10⁵ infectious units, which were further enhanced by repeated immunization. Three doses of 10² infectious units could elicit anti-PSMA responses, further increasing with dose elevation [239]. Moreover, Garcia-Hernandez et al. reported that a treatment regimen, in which a gene gun–delivered PSCA-cDNA as the prime VEEV replicons encoding PSCA as the boost, led to robust immune cell infiltration and prolonged life expectancy in animal prostate tumor models [240]. Besides PSMA and PSCA, the six-transmembrane epithelial antigen of the prostate (STEAP) can also be utilized as an encoding antigen. A VEEV VRP-based STEAP-expressing vaccine candidate induced antigen-specific CD8⁺ T cell responses and elevate the overall survival rate in mice challenged with tumor cells. Furthermore, IFN-γ, TNF-α, and IL-2 secreting CD4⁺ T cells were detected as crucial in tumor rejection (Fig. 8) [241].

Hepatocellular carcinoma

Ranks as the sixth most common cancer and the third leading cause of cancer-related deaths globally, with more than 900,000 new cases and over 830,000 deaths reported in 2020. Virus-related liver diseases are largely connected to hepatocellular carcinoma. Vaccination against hepatitis B and antiviral treatment for hepatitis B and C infections are effective in preventing virus-related hepatocellular carcinoma. [242].

SV40 T immunodominant peptides have been proven to be highly CD8⁺ T cell promotive against hepatocellular carcinoma in preclinical studies and cancer patients. However, only incomplete tumor regression was witnessed. Couty et al. reported tumor environment modulatory effects of mengovirus replicon that enhanced the treatment effect of the peptide vaccine, escalating tumor regression to tumor eradication. This phenomenon was associated with the local recruitment of innate immunity effectors (Fig. 8) [243].

Colorectal cancer

In the US, colorectal cancer is the second leading cause of cancer-related fatalities [244]. The early-onset colorectal cancer, diagnosed in patients under 50 years old, has seen an increase over the years [245].

Hoang-Le et al. designed a KUNV replicon expressing granulocyte colony-stimulating factor (GM-CSF) vaccine, which caused > 50% eradication rate of established subcutaneous CT26 colon carcinoma, and regression of CT26 lung metastasis [246]. Furthermore, combination immunization of IL-12-expressing VEEV VRP and CEA-expressing VEEV VRP greatly enhanced CEA-specific T-cell and antibody responses [247]. Taking advantage of next-generation sequencing, Maine et al. designed a replicon vaccine using the Synthetically Modified Alpha Replicon RNA Technology platform (SMARRT) to encode tumor-specific neoantigens or tumor-associated antigens. This platform includes a downstream loop from Old-World alphaviruses to prevent replicon protein translational shutdown. The SMARRT vaccines successfully induced polyfunctional CD4⁺ and CD8⁺ T cell responses. These immune responses got further enhancement with combination therapy including SMARRT vaccine encoding cytokines and traditional checkpoint inhibitors [248] (Fig. 8).

Malignant mesothelioma

Is an uncommon yet highly aggressive form of cancer that is typically triggered by exposure to asbestos fibers [249]. It causes chest pain, dyspnea, weight loss, night sweats, and pleural effusion in patients. Ninety percent of diagnosed cases are already in the advanced stage, which inevitably leads to death [250].

A fowlpox replicon-based vaccine candidate that encoded survivin was designed by Bertino et al. and was proved to strengthen CD8⁺ T cell infiltration at TME, increase IFN-γ-producing CD8⁺ T cells in both spleen and lymph nodes, delay tumor growth, and improve animal survival (Fig. 8) [251].

Hematolymphoid tumor

Since 1990, incident cases of hematologic malignancies have been on the rise globally, reaching 1,343,850 cases in 2019. However, the age-standardized death rate (ASDR) for all types of hematologic malignancies has been decreasing. In 2019, the ASDR for leukemia, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma were 4.26, 1.42, 3.19, and 0.34 per 100,000 population, respectively, with Hodgkin lymphoma experiencing the most significant decline in death rates [252].

VEEV VRP encoding mouse fms-like tyrosine kinase-3, a receptor tyrosine kinase that participates in the proliferation and survival of hematopoietic stem cells, was shown to break B cell tolerance, which has been troubling immunoglobulin therapies [253]. The immunization of VRP-fms-like tyrosine kinase-3 elicited a rapid fms-like tyrosine kinase-3-specific IgG B cell response in both leukemia and lymphoma mouse models. Furthermore, IgGs against other tumor-associated antigens were also detected [253]. A non-spreading RVFV was designed by deleting genes for NSs and glycoprotein-encoding genome segments. Oreshkova et al., utilized this platform to construct a vaccine expressing a single CD8-restricted epitope, immunodominant NLVPMVATV epitope of human cytomegalovirus (CMV) pp65, which evoked CD8⁺ T cell responses and induced complete clearance of lymphoma cells expressing target antigen (Fig. 8) [254].

Clinical research on replicon vaccines

Clinical research on infectious disease vaccines

There is considerable anticipation regarding the application of replicon vaccines in the field of infectious diseases, attributed to their inherent characteristics (Table 1). The combination of straightforward preparation and self-amplifying properties positions replicon vaccines as promising candidates for addressing a variety of infectious diseases. Henao-Restrepo et al. conducted a ring vaccination cluster-randomized trial in Guinea to test the effectiveness and efficacy of a VSV-based EBOV vaccine that expresses EBOV surface glycoprotein. Contacts and contacts of contacts of diagnosed EBOV disease individuals were recruited in the trial. The vaccine performed 100% efficacy in preventing EBOV disease in immediately vaccinated individuals 10 days or more after vaccination, with no new cases observed among 3775 immediately vaccinated individuals while 23 cases among 4507 delayed or never-vaccinated individuals (PACTR2015030010) [255, 256]. On 29 May 2017, official approval was given for the use of rVSV-ZEBOV by the Democratic Republic of the Congo [257]. Bernstein et al. constructed a bivalent alphavirus replicon particle CMV vaccine that encoded gB and pp65/IE1. In a phase I trial, in 40 healthy volunteers not infected by CMV, the 3-dose bivalent alphavirus vaccination regimen illustrated fine tolerance. Ex vivo experiments suggested the vaccine recipients were able to elicit IFN-γ responses to CMV antigens, neutralizing antibodies, and polyfunctional CD4⁺ and CD8⁺ T cell responses despite different per dose levels (1 × 10⁷ IU/1 × 10⁸ IU) and other administration routes (intramuscular/ subcutaneous) (NCT00439803) [258]. The phase I trial conducted by Wecker et al. investigated the safety and immunogenicity of the alphavirus replicon-based HIV-1 vaccine that encoded a nonmyristoylated form of gag. This trial indicated that despite that the vaccine was well tolerated, the application of it on human HIV-1 patients would be halted due to suboptimal immunogenicity in noninfected healthy individuals, with low levels of binding antibodies and T cell responses even at the highest vaccination dose (1 × 10⁸ IU) (NCT00063778, NCT00097838) [259]. The phase I/II clinical trial conducted by Low et al. tested the VEEV-based full-length SARS-CoV-2 S protein-expressing vaccine candidate (ARCT-021), which was formulated with proprietary LUNAR® LNP, indicated that ARCT-021 was well-tolerated in one 7.5 μg dose and two 5.0 μg doses with slight local solicited adverse events and systemic solicited adverse effects. Further, dose levels of 5.0 μg, 7.5 μg, and 10 μg in a one-dose regimen targeting the young population and dose levels of 3.0 μg and 5.0 μg in a two-dose regimen targeting both young and old populations witnessed 100% seroconversion for anti-S IgG. The titers of neutralization antibodies generally increased with the increasing dose, while the titer reached a steady state after the dose level reached 5.0 μg and higher. Moreover, T-cell responses were also elicited. In both one dose cohort and two dose cohort, younger adults receiving 5.0 μg obtained the largest T cell responses (NCT04480957) [260]. However, another phase I trial held by Pollock et al. investigating the optimal dose level of a VEEV-based LNP-formulated full-length S glycoprotein vaccine (COVAC1) stated that the vaccine failed to evoke 100% seroconversion even at the highest dose level (10.0 μg), which had highest seroconversion rate among all dose levels, 35% after the first vaccination and 61% after the second vaccination. Neutralizing antibody increased with the increase of dose, and stabilized after the dose level reached 5 μg. In the trial, Pollock et al. chose the membrane-tethered trimer form of the full-length S glycoprotein with two proline amino acid substitutions (2P-S, K968P, and V969P), which promoted stabilization of the prefusion conformation (ISRCTN17072692, EudraCT 2020–001646-20) [261]. A phase 2a clinical trial with COVAC1 was implemented by Szubert et al., in which subjects received 1 μg then 10 μg of COVAC1, 14 weeks apart. The modified vaccination regimen induced 80% of seroconversion in SARS-CoV-2 naïve subjects, and the first 1 μg vaccination induced increases in S IgG binding antibodies for most SARS-CoV-2 positive individuals. 56% of SARS-CoV-2 naïve subjects detected neutralizing antibodies after completing the vaccination regimen (ISRCTN17072692, EudraCT 2020–001646-20) [262]. EXG-5003 was a specially designed VEEV-based SARS-CoV-2 vaccine that expressed the RBD of SARS-CoV-2 and functions at the skin temperature but was inactivated at the core temperature. Cooperating with the intradermal injection route, the vaccine deployed DC responses and cellular immunity. The Phase I/II Clinical Trial on the EXG-5003 revealed robust antigen-specific cellular immunity but no increased SARS-CoV-2 RBD antibody titers or neutralizing antibody titers. Further, two prior doses of EXG-5003 enhanced the cellular immune responses induced by the approved mRNA vaccine (BNT162b2 or mRNA-1273), indicating the priming effect of EXG-5003 (NCT04863131) [263]. Besides all these completed clinical trials, phase I clinical trials on influenza replicon vaccine AVX502 (NCT00440362) [264], SARS-CoV-2 replicon vaccine HDT-301 formulated with LION (NCT04844268, NCT05132907) [265, 266], and QTP104 (NCT05876364) [267] was launched with no presented results. Clinical trials of the application of replicon vaccines in infectious diseases indicate a good safety level of this technology in vaccine development. The immunogenicity of replicon vaccines varies, which may be the result of different diseases, different vaccine designs, and different delivery platforms.

Table 1 Published and registered clinical trials of repRNA vaccines against infectious diseases

Clinical research on cancer vaccines

The robust T cell responses witnessed in preclinical studies highlight the possibility of deploying replicon vaccines against cancers (Table 2). In the year 2010, Galanis et al. reported the effects of a recombinant MV replicon expressing CEA on recurrent ovarian cancer patients who were unresponsive to Taxol and platinum and had normal CEA levels. Seven intra-peritoneal doses did not increase anti-measles antibodies or anti-CEA antibodies. Immunosuppression that usually caused by WT MV infection, and viral shedding did not occur after vaccinations. 14/21 patients had disease stabilization, the median duration of which was 92.5 days. Five patients obtained a decrease in CA-125 levels. In addition, the median survival of the vaccinated patients (12.15 months) doubled compared to the recorded statistic of not-treated patients (6 months) (CDR0000515008) [268]. A VEEV VRP-based vaccine expressing CEA (AVX701) has been tested against advanced cancer patients, including colorectal, appendiceal, pancreas, lung, and breast cancers. Rewarding results were recorded in the high dose cohort (4 × 10⁸ IU per dose) as repeated immunizations led to increased CEA-specific CD4⁺ and CD8⁺ T cell responses and antibody responses despite the high titers of vector-specific neutralizing antibodies and Tregs. 2 patients achieved stable disease (SD), 1 reached complete response (CR) and 2 patients with no evident disease remained the condition after completing all vaccinations (NCT00529984) [269]. Crosby et al. reported the follow-up data of NCT00529984, that targeting patients with stage IV cancer, median follow-up was 10.9 years and 5 year survival was 17%. Furthermore, 12 patients with stage III colorectal cancer were enrolled and administered with 4 doses of 4 × 10⁸ IU AVX701 intramuscularly with 3 week intervals. CEA-specific humoral immunity was detected and stage III cancer patients showed elevated CD8⁺ T_EM cell responses and downregulated FOXP3⁺ Tregs. Additionally, CEA-specific, IFN-γ-producing CD8⁺ granzyme B⁺ T_CM cells appeared to be increased after vaccination. The stage III colorectal cancer patients were 100% alive at a median follow-up of 5.8 years, and the recurrence rate was only 25% (NCT01890213) [270]. This clinical outcome is promising since stage III patients treated with 3 months or 6 months of postoperative adjuvant chemotherapy only have 82.4% and 82.8% of 5 year overall survival, respectively, and 69.1% and 70.8% of disease-free survival rates, respectively [271]. The phase I dose escalation trial of a VEEV-based PSMA-VRP vaccine suggested no evident cellular immune response or clinical benefit was induced in either 0.9 × 10⁷ IU or 0.36 × 10⁸ IU groups. Neutralizing antibody titers were elevated after vaccination but were still suboptimal [272]. A VEEV-based VRP-HER2 vaccine designed by Crosby et al. showed a good safety level and induction of HER2-specific T cells and antibodies, while only partial clinical responses were witnessed, one reached partial response (PR), two reached SD. For those patients that gained lengthened progression-free survival, the perforin expressed by memory CD8 cells was regarded as significant. In addition, the antibodies induced participated in both antibody-dependent cellular cytotoxicity and HER2 internalization (NCT01526473) [273]. An active phase II clinical study was launched to investigate whether pembrolizumab could enhance the tumor-infiltrating and peripheral blood immune response against advanced HER2-overexpressing breast cancer treated with a VRP-HER2 vaccine (NCT03632941) [274]. The MV-vectored human sodium-iodide symporter-expressing vaccine was constructed by Dispenzieri et al., the optimum dose of which, TCID50 10¹¹, was detected in phase I and was applied in a phase II trial. These recombinant viral particles were selectively oncolytic to tumor cells expressing the CD46 receptor. RNA replication occurred in gargle, blood, and urine specimens from patients with recurrent or refractory multiple myeloma. After vaccination, one patient achieved CR, and other patients saw transient drops in serum immunoglobulin-free light chains with grade 3–4 adverse effects documented (NCT00450814) [275]. SFV replicon particles that encode HPV16-derived antigens E6 and E7 (Vvax001) were investigated in 12 patients with a history of cervical intraepithelial neoplasia, which were grouped into four cohorts equally to be treated with different dose levels, from 5 × 10⁵ to 2.5 × 10⁸ infectious particles per immunization. Three immunizations with Vvax001 at all dose levels were well-tolerated and elicited CD4⁺ and CD8⁺ T cell responses in all 12 patients (NCT03141463) [276]. Positive results from the Phase I clinical trial supported the recruitment of patients with premalignant cervical lesions for a Phase II clinical trial with Vvax001 (NCT06015854) [277]. Three immunizations with Vvax001 (5 × 10⁷ infectious particles) induced reductions in HPV16-positive cervical intraepithelial neoplasia grade 3 (CIN3) lesion sizes in 17/18 patients, histopathological CR in 50% of patients, HPV16 clearance in 63% of patients with no serious adverse effects and recurrences at present [278]. Clinical trials demonstrate that the replicon vaccines are safe to be utilized against cancers. Some trials claimed positive clinical responses after immunizations, while others were not documented.

Table 2 Published and registered clinical trials of repRNA vaccines against cancer

Conclusion

Studies on repRNA have introduced novel opportunities and challenges in vaccine development. Its high efficacy in expressing GOI at low doses, coupled with the adjuvant properties of viral backbones, significantly enhances vaccine immunogenicity. Furthermore, the nucleic acid nature of repRNA eliminates the need for steps like gene cloning, cell culture, and protein purification, thereby expediting vaccine design and production while also reducing associated costs. This significantly aids the vaccine industry in addressing outbreaks of infectious diseases, as evidenced by the extensive research conducted on SARS-CoV-2 replicon vaccines during the COVID-19 pandemic. Besides, replicons hold great potential in gene therapy, the development of multivalent vaccines, and protein replacement therapies, etc. Alphaviruses have emerged as the predominant viral vectors utilized in replicon vaccine design. It is imperative to explore a broader array of viral vectors to assess their feasibility and identify appropriate contexts for their application. Additionally, the choice of delivery vehicle and route of administration can significantly impact immune responses and the outcomes of immunizations. Continuous investigation into synthetic replicon carriers that target specific cell populations and enhance vaccine efficacy is warranted. Furthermore, replicon vaccines may be employed independently or in conjunction with other therapeutic agents. A comprehensive and meticulous refinement of vaccination strategies is essential to optimize their efficacy. Replicon RNA represents a promising yet insufficiently explored technology in the realm of vaccine development, offering potential opportunities for combating infectious diseases and other malignancies such as cancers.