Insights into Antibody-Mediated Alphavirus Immunity and Vaccine Development Landscape

Alphaviruses are mosquito-borne pathogens distributed worldwide in tropical and temperate areas causing a wide range of symptoms ranging from inflammatory arthritis-like manifestations to the induction of encephalitis in humans. Historically, large outbreaks in susceptible populations have been recorded followed by the development of protective long-lasting antibody responses suggesting a potential advantageous role for a vaccine. Although the current understanding of alphavirus antibody-mediated immunity has been mainly gathered in natural and experimental settings of chikungunya virus (CHIKV) infection, little is known about the humoral responses triggered by other emerging alphaviruses. This knowledge is needed to improve serology-based diagnostic tests and the development of highly effective cross-protective vaccines. Here, we review the role of antibody-mediated immunity upon arthritogenic and neurotropic alphavirus infections, and the current research efforts for the development of vaccines as a tool to control future alphavirus outbreaks.


Introduction
Mosquito-borne alphaviruses are Group IV viruses that belong to the family Togaviridae [1]. They are enveloped, positive-sense, single-stranded RNA viruses with a size of ≈70 nm bearing a ≈11.7 kilobases genome which encodes four non-structural proteins (nsP1, nsP2, nsP3 and nsP4) that serve as the virus' replication machinery, and five structural proteins (capsid, E3, E2, 6K and E1) that participate in the envelope assembly process [1]. Clinically, alphavirus infections in humans results in the development of viremia followed by an onset of febrile symptoms [2]. The development of inflammatory conditions compromising joints and muscle tissues has been associated to arthritogenic alphaviruses such as chikungunya virus (CHIKV), O'nyong nyong virus (ONNV), Mayaro virus (MAYV), Ross River virus (RRV), Semliki Forest virus (SFV) and Sindbis virus (SINV) with records of persistent polyarthralgia in a fraction of patients. Conversely, neurotropic alphaviruses such as Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalitis virus (WEEV) and Venezuelan Equine Encephalitis virus (EEEV) have been linked to the induction of lethal encephalitis in humans and animals [3,4].
Historically, alphaviruses have a proven record of causing massive outbreaks in susceptible populations [5][6][7][8]. Additionally, the appearance of mutations favoring their ecological fit to new vectors has fueled alphavirus propagation worldwide [9,10]. A clear example of their potential as a health threat is the re-emergence of CHIKV in 2004 after a hiatus of more than 50 years since its discovery [5]. More recently, tropical emerging alphaviruses such as ONNV and MAYV are believed to have the potential to become future major epidemics [11][12][13]. This is due, in part, to the lack of robust diagnostic tests to differentiate alphavirus infections from other febrile tropical diseases and the absence of continuous epidemiological surveillance masking their real potential for spread beyond endemic areas [14][15][16].
Although alphavirus infections are generally not life threating the economic and social costs incurred during outbreaks are thought to be high [17][18][19]. Moreover, the lack of approved treatments leaves management of alphavirus infections to supportive care [20]. Interestingly, a body of work suggests that the alphavirus infection triggers potent humoral responses in exposed populations which seem to confer protection against re-infection [21]. Therefore, a better understanding of the antibody responses against alphaviruses is crucial for the development of vaccines, which would represent a big advantage in the control of alphavirus infections.

Virus-Specific Antibody Kinetics Upon Natural Infection with Alphaviruses
The current knowledge on the role of antibody-mediated immunity upon viral infection has been gathered from cohort studies following major alphavirus outbreaks. Serological surveys following CHIKV re-emergence in 2004 reported the quick development of IgM responses between five to seven days post-illness onset (PIO) [22,23]. IgM is generally detectable for up to three months post-infection [24][25][26]. However, long-lasting IgM has been often reported in patients with long-term CHIKV-induced polyarthralgia, which might indicate a constant antigenic stimulation due to viral persistence [27]. After the initial detection of IgM antibodies, IgG seroconversion reportedly occurs between 4 to 10 days PIO taking over as the main immunoglobulin detected in serum [22,28]. Notably, IgG3 antibodies become the dominant IgG subtype produced upon infection and have been associated to efficient viral clearance and protection against chronic CHIKV symptoms [23]. Importantly, IgG responses persist for several years and might be potentially lifelong [29].
ONNV and MAYV, both closely-related to CHIKV, are re-emerging arthritogenic alphaviruses believed to be confined to sub-Saharan Africa, and Latin America, respectively [6,11,12,15]. Following the largest ONNV outbreak in Uganda involving more than two million cases between 1959-1962 [6,30], the induction of potent neutralizing antibodies was described [31]. The first study cohort that evaluated IgM kinetics upon ONNV infection in Uganda [32] reported the appearance of IgM antibodies during the second week PIO which remained elevated for two months. In contrast, reports from imported ONNV cases in Europe described detectable IgM levels as early as five days PIO [33,34]. ONNV-specific IgG levels are increased in serum after the third week and remain high beyond two months PIO [11,34]. However, whether IgG responses are long-lasting remains unknown. Similarly, endemic MAYV infections are characterized by the early appearance of IgM antibodies (3-8 days PIO) that might last for one to three months [35,36]. IgG becomes detectable around 4-10 days PIO [35] and remains elevated after 6-12 months [37,38]. Interestingly, unlike ONNV and CHIKV infections, persistent arthralgia has been reported in more than half of MAYV-infected individuals and although MAYV-specific antibody responses are critical for disease resolution it is seemingly insufficient to protect patients from the development of chronic joint manifestations [39].
Other alphaviruses linked to continuous small outbreaks associated with arthritic manifestations in human populations are RRV and SINV. RRV is endemic to Australia and is responsible for approximately 4000-5000 cases annually [40]. Typically, antibody kinetics upon RRV infection are characterized by the development of IgM titers between 7-10 days PIO, peaking at two to three weeks and lasting for 1-3 months [41,42]. IgM response rapidly declines after three weeks PIO as IgG becomes dominant [42,43]. Interestingly, IgM persistence has been reported in some RRV cohorts [41]. In one study [44], 19/116 (16.4%) of participants had detectable IgM levels that lasted between seven months to eight years PIO. Likewise, less prevalent SINV has also been linked to the development of persistent virus-specific IgM levels. Although, generally, the antibody response upon SINV infection generates IgM antibodies after 6-9 days PIO and IgG antibodies after 9-14 days, some reports described the presence of detectable IgM levels up to four years suggesting active viral replication [45][46][47]. The clinical relevance of persistent IgM levels following RRV and SINV infection is yet to be determined.
Neurotropic alphaviruses such as EEEV, WEEV and VEEV cause sporadic cases of human encephalitis in the Americas [4]. While the natural reservoirs for these viruses are primarily birds and equines, humans are susceptible to infection when the enzootic cycle of transmission leaks into mosquito populations with a wide range of hosts [48]. Given that human cases are rare, there is a lack of information regarding the development of antibody responses upon natural infections by neurotropic alphaviruses. In a paired serology study [49], virus-specific antibody responses were profiled in a cohort of 20 EEEV and 17 WEEV-infected patients. IgM antibodies were observed as early as 1 PIO, peaking after 1-2 weeks and remaining detectable for up to three months. In contrast, IgG responses appeared during the second week PIO and remained elevated until the end of the follow-up period.

Experimental Evidence of the Role of Antibodies in Alphavirus Immunity
To better understand the role of antibody-mediated immunity upon alphavirus infection, several animal models have been used allowing the detailed examination of the cellular compartments responsible for the initiation of humoral immunity. The role of B cells in alphavirus immunity has been described in experimental CHIKV infections. Inoculation of µMT mice (lacking mature B cells) with CHIKV resulted in higher viremia that persisted up to 402 days post-infection (DPI). In contrast, infected wild type (WT) mice were able to control the virus during the second week post-inoculation [50]. Similar findings were reported in other studies, where mouse strains lacking B cells (µMT, Rag1, Rag2/IL2rg, NRG) infected with CHIKV displayed increased and persistent viremia for up to 515 DPI [51,52].
B cells also play an important role in alphavirus-induced encephalitis. Although SINV infections in humans are known to cause arthritic manifestations, SINV has been frequently used as a model of alphavirus-induced encephalomyelitis in adult immunocompetent mice given the virus ability to infect neurons [53]. Intracerebral inoculation of SINV in µMT and severe combined immunodeficiency (SCID) mice resulted in defective viral clearance from the brain, brain stem and lumbar spinal cord, virus persistence and recrudescence compared to WT mice [54]. The individual contributions of IgM and IgG antibodies to SINV clearance from brain tissues were assessed in another study [55] where infection in AID −/− (unable to produce IgG), sIgM −/− (unable to produce IgM) and AID −/− sIgM −/− double-knockout mice resulted only in AID −/− sIgM −/− being unable to control infection efficiently suggesting that either IgM or IgG antibodies are sufficient to clear SINV from the central nervous system (CNS). Similar results were obtained in SFV models of encephalitis where infection of µMT [56], SCID [57] and nude mice with impaired antibody switching [58] led to viral persistence.
Infiltrating virus-specific B cells were observed in infected tissues in a murine model of SINV-induced encephalitis [59,60]. Following intracranial virus inoculation, expansion of IgM-secreting plasmablasts was reported in the cervical lymph nodes. Infiltration of CD19+ B cells occurred between 3-7 DPI and coincided with the starting of viral clearance. During the clearance of persistent viral RNA (from 8-80 DPI), the accumulation of SINV-specific IgG and IgA-secreting B cells was observed being associated with increased SINV antibody titers over time [60]. In a subsequent study, it was reported that the brain microenvironment during the early stages of SINV infection facilitates the migration, differentiation, expansion and long term survival of SINV-specific B cells [59].
Follicular helper T cells (T FH ) are a subset of CD4 T cells involved in the activation of B lymphocytes and the establishment of robust antibody responses following antigen stimulation. T FH promotes B cell differentiation, isotype switching and affinity maturation. In experimental CHIKV infections, the use of CD4-deficient mice ruled out the role of CD4 T cells in viral clearance from infected tissues [61]. However, one study demonstrated impaired IgM and IgG (IgG2c, IgG1, and IgG2b) production in mice lacking CD4 T cells following CHIKV inoculation [62]. Albeit reduced virus-specific antibody levels, the neutralizing capacity of sera from virus-infected CD4-deficient mice was marginally affected [62]. Likewise, another study showed similar results upon CHIKV inoculation of MHCII ∆/∆ mice (defective of T FH ) [51]. MHCII ∆/∆ animals were unable to generate IgG1 antibodies and produced ≈100 fold lower IgG2c levels than WT controls. Nonetheless, MHCII ∆/∆ mice were still able to control virus infection [51]. The generation of virus-specific neutralizing antibodies in MHCII ∆/∆ mice suggests a T-cell independent B cell activation characterized by the inability to generate memory B cells. Whether CHIKV-specific antibody responses in mice lacking CD4 T cells are long-lasting remains to be elucidated.

Viral Antigenic Regions Targeted by Neutralizing Antibodies
The notion of targeting humoral immunity as a therapy against alphavirus infection has been investigated since the late 1930s following the isolation of EEEV, WEEV and VEEV. In a series of seminal studies involving immunization of guinea pigs [63][64][65][66], the subcutaneous inoculation of live EEEV and WEEV strains protected guinea pigs from lethal intracranial infection [63]. Additionally, it was observed that immunization with formalin-inactivated virus strains induced the production of neutralizing antibodies at a comparable level than animals immunized with live viruses [64][65][66]. Subsequent studies reported that passive transfer of hyperimmune rabbit serum protected mice, guinea pigs and rabbits from WEEV infection [66,67]. Similarly, passive serum transfer was shown to be effective at protecting mice from the development of neurological complications upon infection with a neuroadapted strain of SINV [68,69]. Comparable observations were reported in experimental infection models of VEEV [70], CHIKV [71,72], RRV [73] and SFV [74].
The first attempts in identifying the exact structural regions, recognized by most neutralizing antibodies produced upon infection, were conducted in experimental infection models of alphavirus encephalitis. Structurally, the envelope of an alphavirus virion has a T = 4 icosahedral symmetry [75]. E1 and E2 are two envelop surface glycoproteins exposed in the viral spike as a heterodimer [75] (Figure 1). It is believed that the E1-E2 heterodimer interacts with host receptors thus mediating viral entry [75]. Additionally, the E1 and E2 glycoproteins were postulated as highly immunogenic regions since their location in the spike facilitates antigenic recognition. In line with this, early works mapped antigenic sites involved in VEEV, SINV and SFV neutralization to the E1 and E2 proteins using competitive binding assays but the exact amino acid sequences were not determined [76][77][78]. Later, a major antigenic region involving three epitopes important in the neutralization of RRV was identified in the E2 protein (incorporating residues 216, 232 and 234) [79]. Similarly, analysis of antibody escape variants determined important antigenic regions between amino acids 181 and 216 on the E2 protein of SINV [80]. A major neutralization domain was also identified between residues 182-207 for VEEV [81].
Following CHIKV reemergence in 2004 several reports identified major linear antigenic sites in the CHIKV E2 protein that induced the production of potent neutralizing antibodies. Using a CHIKV proteome-wide screening approach, a single linear peptide located at the N-terminus of the E2 glycoprotein, E2EP3, was reported as strongly recognized by convalescent CHIKV patients from different cohorts [23]. Furthermore, experimental CHIKV infection in mice and non-human primates (NHP) validated E2EP3 as an immunodominant linear epitope inducing potent neutralizing antibodies [23,62,82]. Interestingly, mice immunization with E2EP3 alone reduced joint swelling and viremia upon CHIKV challenge [23]. In another study focusing on human antibody responses to SINV in cohort from Finland, 6 linear epitopes, located in the capsid, E2, E1 and PE2 (uncleaved E3-E2) proteins, were reported [83]. Three of these epitopes were located to the glycoprotein spike complex between the residues 209-226 of E1 (E1-P5), 273-290 (E2-P3) and 308-325 (E2-P4) of E2 [83]. Interestingly, the E2EP3 equivalent of SINV remained non-reactive suggesting that antibody kinetics against linear E2EP3 between populations exposed to CHIKV and SINV might differ [83].  Figure S1). The development of mouse and human monoclonal antibodies against different alphaviruses helped further the understanding of antigenic responses upon infection by the identification of conformational epitopes. Early works have shown the therapeutic value of mouse monoclonal antibodies in models of alphavirus encephalitis by SINV [84][85][86][87][88], SFV [56,57,89] and VEEV [78]. Interestingly, it was observed that neutralizing monoclonal antibodies target antigenic regions in the E2 protein. Whereas, non-neutralizing antibodies bind to the E1 protein, yet both are able to confer protection upon alphavirus infection, thereby suggesting other mechanisms of protection in vivo besides virus neutralization [48]. Several monoclonal antibodies targeting both E1 and E2 proteins have been reported in the context of arthritogenic alphavirus infection. Mouse monoclonal antibodies targeting the A and B domain of E2 and the domain II of E1 [90][91][92] and the capsid protein [93,94] have been reported for CHIKV. Likewise, human anti-CHIKV monoclonal antibodies were found to target conformation epitopes in the E2 glycoprotein A (containing a putative RBD [95]) and B (shielding the fusion loop in E1 [96]) domains and proved therapeutic value in experimental NHP infections [90,97,98]. Monoclonal antibodies recognizing epitopes predominantly between residues 58-80 (domains A) or residues 180-215 (domain B) of the E2 glycoprotein have been also reported in the context of SINV [83], VEEV [81], EEEV [99][100][101], RRV [102] and MAYV [103].
The combined evidence suggested the existence of common antigenic sites in the viral spike across alphaviruses, particularly in the E2 protein. These sites are likely required for interaction with host cell receptors suggesting that antibody binding might inhibit infection during viral attachment, entry, fusion or egress [90]. In line with this, a recent study reported the discovery of Mxra8, a cell adhesion molecule, as a host receptor required for viral entry of multiple arthritogenic alphaviruses [104]. Genetically altering mouse or human Mxra8 resulted in diminished infection, conversely, overexpression of Mxra8 in cell lines increased infection rates by CHIKV, ONNV, MAYV and RRV [104,105]. Interestingly, mutagenesis experiments suggested E2 domains A and B as the putative binding site for Mxra8 [104]. This notion was later confirmed by cryo-electron microscopy images of Mxra8 bound to CHIKV [106,107]. Mxra8 sits onto a cleft formed by two contiguous CHIKV E2-E1 heterodimers in one trimeric spike while engaging a neighboring spike [106]. It is believed that this interaction works against the virus by obstructing viral fusion [106]. Importantly, human neutralizing antibodies that recognize regions of the A domain of E2 inhibited the binding of Mxra8 supporting the interactions determined in the cryo-EM atomic model. Notably, Mxra8 seems to not be a receptor for neurotropic alphaviruses [104]. The alignment of CHIKV residues involved in Mxra8 binding reveled a degree of conservation in arthritogenic alphaviruses (44%), but diverged from neurotropic Alphaviruses (14%) which might explain the negative results in the context of SINV, EEEV, WEEV and VEEV infections [106]. In summary, the characterization of alphavirus antigenic epitopes has proven beneficial to pave the way for the development of antibody therapies and vaccines.

Alphavirus Vaccine Development
Recent decades have seen increased rates of geographic dispersal of arboviral reemergence, due to factors such as growth of global transportation, urbanization and failure of mosquito control [108][109][110][111]. Given that humans appear to be the only amplification hosts and viral reservoir during urban transmission [112,113], another effective means of controlling the spread of infection is through vaccination. While there are currently no licensed or approved vaccines available for alphaviruses, a multitude of approaches have been used to develop vaccine candidates capable of, not only generating high levels of antibodies, but also providing long-lasting protection, with the ease of administration and production requirements. Multiple methods such as live-attenuated viruses, inactivated viruses, virus-like particles (VLP), recombinant subunit vaccines and chimeric vaccines have been explored for vaccine options ( Figure 2 and Table 1).

Live-Attenuated Vaccines
With the development of alphaviruses in reverse genetic systems, more research has been focused on the rational design of live-attenuated vaccines [221,222] in overcoming potential issues, such as genetic reversion mutations in vaccines [223,224], with highly specific mutations or alterations of the original parental virus genome. In addition, not only are the safety profiles of these vaccines is greatly improved, protection with a only single dose is also achieved [225].
An engineered live-attenuated option for alphavirus vaccine design involves the rational design of downregulating the expression of particular structural proteins with the introduction of a picornavirus (encephalomyocarditis virus) internal ribosome entry site (IRES) into the viral genome. For example, this is demonstrated in a VEEV vaccine candidate, ZPC/IRES, where the expression of the capsid protein is minimalized by translocating its gene to a separate opening reading frame downstream of the envelope glycoprotein genes and interrupting its expression with the introduction of a IRES [114,226]. However, the highly immunogenic envelope glycoproteins E3-E1 were not manipulated, but the insertion of IRES into the genome would functionally alter the host range as replication of the live virus is restricted in mosquitoes.
ZPC/IRES is based on a full-length clone of a wild type VEEV subtype ID from Zulia state, Venezuela from a sentinel hamster exposed in a tropical lowland. CD-1 mice immunized with 10 5 PFU of ZPC/IRES developed strongly neutralizing antibodies, with PRNT80 of average reciprocal titer of 324 by 20 weeks post immunization. Subsequently, when immunized mice were challenged with the lethal VEEV subtype IC strain 3908 (10 5 PFU, subcutaneous or 10 4 PFU, aerosol route) 4 weeks after immunization, all mice retained their weight and failed to show any signs of disease and survived, compared to mock-vaccinated mice which succumbed to the lethal infection. Additionally, the study tested the vaccine in a NHP immunization-challenge model in the same study. Vaccinated NHPs had PRNT80 values of 160 to 320, and all vaccinated NHPs were protected against viremia upon challenge with VEEV 3908 strain. Using the VEEV ID strain ZPC738 as the vaccine backbone, which is closely related to subtypes IAB and IC, the authors had aimed to develop an IRES-based, live-attenuated vaccine candidate that could possibly protect against other subtypes of VEEV, given that previous attempts to create a vaccine candidate based on the VEEV subtype IAB V3526 vaccine could not significantly protect against aerosol challenge with a subtype IE VEEV strain [226]. Nonetheless, this hypothesis was not pursued in the study, and it would have been curious to learn whether the ZPC/IRES-immunized animals would be protected from a lethal challenge with VEEV of other subtypes, such as subtypes IA/B and IE.

Inactivated Vaccines
The inactivated Ross River virus (RRV) vaccine is the most developed and advanced vaccine candidate, having been rigorously tested in both preclinical and up to Phase 3 clinical trials. The Vero cell culture-derived whole-virus RRV vaccine was first produced from a viral seed derived from an RRV isolate from a serologically confirmed case of RRV disease in Queensland, Australia, and subsequently inactivated by sequential formalin and UV light treatment after harvest [227]. In pre-clinical testing of the RRV vaccine, CD-1 mice were given two doses of the inactivated RRV vaccines at different experimental doses 28 days apart, without the use of an adjuvant in its formulation. Upon challenge with 10 6 TCID50 of the mouse-virulent RRV prototype strain T48 at 42 days post immunization, a vaccine dose beyond 0.625 µg provided almost complete protection against viremia development at 1-day post challenge. Interestingly, the possible antibody-dependent enhancement by RRV vaccination by a closely related alphavirus infection was investigated, where viremia in CHIKV LR2006 OPY-1-infected-RRV vaccinated mice was significantly reduced as compared to the control. In this heterologous situation, partial cross protection was observed, but the presence of sub-protective levels of RRV vaccine-induced antibodies prevented the enhancement of CHIKV replication [73].
Subsequently, a randomized Phase 3 clinical trial for the RRV vaccine was conducted in Australia to investigate the safety and immunogenicity of the vaccine in a large cohort of 1755 healthy younger adults aged 16 to 59 years and 209 healthy older adults aged > 60 years [115]. The 2.5 µg Al(OH)3-adjuvanted vaccine was given over three doses (subsequent boosts at 3 weeks and 6 months). The majority of participants in the younger and older adult populations had seroprotective uNT titers after three immunizations with the whole-virus RRV vaccine, and titers of serum IgG antibodies after three immunizations were higher than the serological IgG ELISA titer threshold associated with protection after natural infection with RRV [115]. While the RRV vaccine had been brought forward to Phase 3 clinical trials, and despite the vaccine demonstrating safety and efficacy, it was not considered financially viable to manufacture, despite Queensland recording its largest and worst epidemic between 2014 to 2015 [228,229]. In addition, given that the cost of vaccine trials is hard to justify for a disease that occurs only in Australia and Papua New Guinea, and where the disease is never fatal, efforts to further develop the RRV vaccine were unfortunately halted.

Virus-Like Particles (VLPs)
The VRC-CHKVLP059-00-VP is one of the first potential new CHIKV vaccines to reach advanced development with human clinical testing [116,117]. The CHIKV envelope gene cassette encoding the native polypeptide, E3-E2-6K-E1, of CHIKV strains 37,997 (West African genotype) and LR2006 OPY-1 were inserted into a cytomegalovirus CMV/R expression vector and subsequently transfected into 293T human kidney cells [118]. The resulting VLP product is a CHIKV VLP that is structurally identical to its infectious counterpart (given that structural genes are intact), but is not infectious as its genetic material is removed. While the CHIKV 37997 strain yielded approximately 100 times more VLPs than that from strain LR2006 OPY-1, the former strain was subsequently used to produce the VLPs. Nonetheless, given that the ECSA lineage was responsible for the ongoing outbreak at the time of development, the high degree of amino acid similarity between the two CHIKV strains suggested that the vaccine would be protective against viruses of other genotypes. However, it would have been curious to characterize a VLP produced from a CHIKV strain of the ECSA lineage, given that it is the strain responsible for recent Chikungunya epidemics all around the world [119][120][121][122][123].
BALB/c mice immunized with two doses of 19 µg of CHIKV VLPs intramuscularly generated the highest neutralizing titer against both the homologous strain 37,997 and the heterologous strain LR2006 OPY-1. In addition, NHPs immunized with 20 µg of VLPs developed substantial neutralizing activity to both homologous and heterologous strains after primary immunization. Interestingly, even though the VLP was made from CHIKV 27,997 strain, there was slightly better neutralization of LR2006 OPY-1 compared to 37,997 in both mice and NHPs. The study speculated that this is suggestive that the LR2006 OPY-1 virus may present a conserved epitope to the immune system better than the 37,997 virus. When total IgG antibodies were passively transferred from immunized NHPs to defective type 1 interferon signaling immunodeficient mice (Ifnar1 −/− ), these recipient mice did not develop detectable viremia and all survived a lethal challenge with CHIKV LR2006 OPY-1. This indicated that the humoral immune responses induced by the CHIKV VLPs confer protection against CHIKV infection [124].
This promising data eventually led to further testing in clinical trials-phase 2 studies were concluded and reported in 2020 [117]. The randomized phase 2 clinical trial included 400 healthy adults in outpatient clinics in 6 countries in the Caribbean. Two doses of 20 µg of CHIKV VLP, termed as VRC-CHKVLP059-00-VP in clinical trials, were administered 28 days apart via intramuscular injection. Vaccine-induced humoral immune responses in individuals were comparable with titers from participants vaccinated in the phase 1 trial [116], and serum collected from participants in the phase 1 trial induced neutralizing antibodies against all 3 genotypes of CHIKV. Interestingly, while the phase 2 trial aimed to only enroll CHIKV seronegative participants, 20% of the cohort (in particular, participants from 2 study sites-Dominican Republic and Haiti) were retrospectively found to be seropositive at baseline on the day of the study enrolment, possibly due to seroconversion between screening and study enrolment. A post-hoc analysis demonstrated that the VLP was immunogenic among these seropositive recipients, but a significant difference was observed between the geometric mean ratio between seropositive and seronegative vaccine recipients. Further studies on this specific group of participants to understand the possible effects of seropositivity and efficacy or protection of the CHIKV VLP administered will be interesting, rendering the need for additional clinical trials [117].

Chimeric Viruses
Another option in vaccine development in providing high levels of immunity is the use of a virus-vector system that utilizes an avirulent backbone, but incorporates the expression of viral genetic elements, such as the chimeric vector system for producing foreign gene products.
The insect-only host-restricted Eilat virus (EILV) has recently also been utilized as a chimeric backbone to replace the structural open reading frame with that of EEEV, VEEV or CHIKV [125]. Given that EILV is unable to replicate in vertebrate cells and in brain tissues of infant mice, this enhances the safety aspect of the vaccine, and thus, also serves as a inactivated vaccine, which enhances the expression of particular immunogenic proteins. Separately, the monovalent EILV/EEEV and EILV/VEEV vaccines were efficacious in their protection against lethal alphavirus challenge-immunized CD-1 mice had a high seroconversion rate observed post vaccination and were highly protected from lethal EEEV-FL93 or VEEV-3908 challenge. Compared to mock-vaccinated animals, EILV/EEEV or EILV/VEEV immunized animals had little or no weight loss and were protected from disease. More importantly, a trivalent vaccine containing the EILV/EEEV, EILV/VEEV and EILV/CHIKV chimeras was formulated and assessed if the vaccine could provide protection against lethal challenge with multiple alphaviruses. A single trivalent dose of EILV/VEEV, EILV/EEEV, and EILV/CHIKV elicited neutralizing antibodies against all three viruses and provided >80% protection against VEEV and EEEV lethal challenge. Collectively, this work showed safety combined with strong immunogenicity and ease of production, making the use of the EILV alphavirus chimeric vaccine platform promising and attractive [125]. The use of a trivalent vaccine candidate also serves as a proof-of-concept to show practicality and increases its potential as a vaccine against neurotropic alphaviruses.

Nucleic Acid-Based Vaccines
Commonly known as the 'third-generation vaccine', RNA and DNA vaccines form one of the latest vaccine approaches for alphaviruses. The risk of infection from receiving a vaccine is minimal, given the safety associated with the nucleic acid product [230]. In addition, as some vaccines have been shown to have poor immunogenicity due to the lack of uptake or the need for adjuvants [231], much research over the past decades have explored the design of constructs and novel delivery technologies to overcome these issues. In order to overcome several issues related to traditional vaccine development, such as high cost and difficulty in production, RNA has emerged as an effective platform to deliver vaccines using nanoparticle delivery vehicles, such as liposomes [232][233][234].
A RNA vaccine against CHIKV involves the delivery of the self-replicating RNA genome of the live attenuated CHIKV-NoLS virus with CAF01 liposomes [126]. The mutation in the nucleolar localization sequence (NoLS) in the capsid protein of CHIK-NoLS was previously shown to significantly attenuate viral replication [127]. In the same study, C57/BL6 mice immunized with one dose of CHIKV-NoLS were fully protected from CHIKV infection [127]. In immunodeficient AG129 mice, a single dose of CHIKV-NoLS RNA delivered with CAF01 generated CHIKV-specific neutralizing antibodies. While these immunized AG129 mice developed disease signs, they eventually recover from the immunization, compared to mock-immunized mice. Importantly, CHIKV-NoLS CAF01immunized AG129 mice survive from subsequent CHIKV challenge and do not develop CHIKV-induced footpad swelling or disease. On the other hand, in immunocompetent C57/BL6 mice, CHIKV-NoLS CAF01-immunized mice developed delayed viremia at a similar titer compared to CHIKV-WT, and were protected from footpad swelling. However, immunization with either CHIKV-NoLS CAF01 or CHIKV-NoLS RNA produced significantly lower levels of neutralizing antibody compared to CHIKV-WT inoculation [126]. However, this study showed that the RNA-launched self-assembling viral particles generated immunity and protection that were just as strong as those of wild-type viral particles, suggesting the significant potential of this approach.
While multiple novel approaches have been explored to develop vaccines against alphaviruses, a potential prophylactic strategy that could be the development of a multivalent alphavirus vaccine given the reports of cross-neutralizing antibodies against conserved epitopes in the E2 protein across closely related alphaviruses. This approach would prove useful in endemic areas where alphavirus co-circulation occurs.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.