2.1. 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.
2.2. 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
Δ/Δ mice (defective of T
FH)
[51]. MHCII
Δ/Δ animals were unable to generate IgG1 antibodies and produced ≈100 fold lower IgG2c levels than WT controls. Nonetheless, MHCII
Δ/Δ mice were still able to control virus infection
[51]. The generation of virus-specific neutralizing antibodies in MHCII
Δ/Δ 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.
2.3. 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] (). 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].
Figure 1. Structure of the alphavirus E1-E2 heterodimer. Ribbon diagram (PDB: 3N41) highlighting (A) E1 glycoprotein (domain I: red, domain II: yellow, domain III: blue, fusion loop FL: orange, E2: grey) and (B) E2 glycoprotein (domain A: cyan, domain B: green, domain C: pink, beta-ribbons: purple, E1: grey). (C) Table summarizing reported antibody binding regions in the E1 and E2 glycoproteins of arthrogenic and neurotropic alphaviruses. Numbers in the table refer to in-text citations describing such binding sites (See Reference list). Background color matches protein doimains depicted in (A) and (B). To assess for the degree of conservation among common antigenic regions across alphaviruses a sequence aligment analysis was conducted.
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].
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.