Table of Contents

    Topic review

    Antibody-Mediated Alphavirus Immunity

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    Submitted by: Lisa F.P. Ng

    Definition

    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.

    1. 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.

    2. Antibody-Mediated Alphavirus Immunity

    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 (TFH) are a subset of CD4 T cells involved in the activation of B lymphocytes and the establishment of robust antibody responses following antigen stimulation. TFH 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 MHCIIΔ/Δ mice (defective of TFH) [51]. MHCIIΔ/Δ animals were unable to generate IgG1 antibodies and produced ≈100 fold lower IgG2c levels than WT controls. Nonetheless, MHCIIΔ/Δ mice were still able to control virus infection [51]. The generation of virus-specific neutralizing antibodies in MHCIIΔ/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] (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].
    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.

    3. Alphavirus Vaccine Development

    Recent decades have seen increased rates of geographic dispersal of arboviral re-emergence, 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).
    Figure 2. An outline of the current vaccine options against arthritogenic (left panel) and neurotropic (right panel) alphaviruses. Most of these vaccine candidates are currently under preclinical testing (early preclinical—vaccine candidates tested in mouse models; late preclinical—vaccine candidates currently under testing in non-human primates (NHP)), while a minority of them are currently undergoing clinical trials (Phase 1, 2 or 3). LAV; live-attenuated virus; VLP, virus-like particle; SIN, Sindbis virus; ISFV, Isfahan virus; May, Mayaro virus; EILV, Eilat virus, VSV/VSIV, vesicular stomatitis virus; MV, measles virus; MVA, modified vaccinia virus Ankara. Data curated from literature reported through February 2021. Numbers in superscript refer to reference numbers (See Reference list [23][73][114][115][116][117][118][119][120][121][122][123][124][125][126][127][128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143][144][145][146][147][148][149][150][151][152][153][154][155][156][157][158][159][160][161][162][163][164][165][166][167][168][169][170][171][172][173][174][175][176][177][178][179][180][181][182][183][184][185][186][187][188][189][190][191][192][193][194][195][196][197][198][199][200][201][202][203][204][205][206][207][208][209][210][211][212][213][214][215][216][217][218][219][220]).
    Table 1. List of vaccine candidates against relevant alphaviruses currently under development 1.

    Vaccine Against Virus

    Name

    Strain Vaccine Modelled After

    Phase

    Immunization

    Challenge

    Humoral Immune Response(s)

    Ref

    Dose

    Route

    Schedule

    Dose (Strain, Genotype)

    Route

    Live-attenuated

                     

    CHIKV, ONNV

    RH-CHIKV

    EV-CHIKV

    RHEV-CHIKV

    LR2006 OPY1

    C57BL/6 mice, 3 week old

    106 PFU

    s.c. in the ventral side of the right hind footpad

    Single dose

    106 PFU LR2006 OPY1 or WT-ONNV IMTSSA/5163, 3 mpim

    s.c. in the ventral side of the right hind footpad

    IC50, 613 (RH-CHIKV), 3407 (EV-CHIKV), 921 (RHEV-CHIKV)

    [130]

    CHIKV

    Δ5nsP3 (VLA1553-301 in clinical trials) and Δ6K

    LR2006 OPY1

    C57BL/6 mice, 5 to 6 week old

    104 or 105 PFU

    s.c. in both flanks

    Single dose

    106 PFU LR2006 OPY1, 7 wpim

    s.c.

    NT50, 100 to 1000

    [131][132][133]

    Cynomolgus macaques, 3–4 years old

    105 PFU

    s.c. in the right upper back side

    Single dose

    100 AID50 (corresponding to 7000–10,000 PFU) LR2006 OPY1, 123 dpim

    i.v.

    NT50, >1000

    Human clinical trial, Phase 1

    3.2 × 103,

    3.2 × 104 or 3.2 × 105 TCID50

    i.m.

    Two doses (0 and 6 months, or 0 and 12 months)

    NA

    NA

    GMT, 592.6 to 686.9

    CHIKV

    CHIKV-NoLS

    LR2006 OPY1

    C57BL/6 mice, 21 days of age

    104 PFU

    s.c.

    Single dose

    104 PFU of LR2006 OPY1 or Ross River virus, 30 dpim

    s.c.

    <10% cells infected at 10-1 serum dilution

    [127]

    CHIKV

    Stop CHIKV

    SuperStop CHIKV

    LR2006 OPY1

    C57BL/6 mice, 5 week old

    104 PFU

    s.c.

    Single dose

    ND

    ND

    ~5–25 (Stop CHIKV) and ~10–25 (SuperStop CHIKV) fold reduction compared to mock

    [134]

    CHIKV

    ChikV HR

    37997

    C57BL/6 mice, 28 days of age

    ∼103 PFU

    s.c. into the left footpad

    Single dose

    103 PFU CHIKV SL15649, 28 dpim

    s.c. in the footpad

    PRNT50, 5 to ~500

    [135]

    CHIKV

    Heparin sulfate cell culture adapted

    LR2006 OPY1

    CD-1 mice, 21 days old

    105 GE

    s.c. in the rear footpad

    Single dose

    103 PFU LR2006 OPY1, 21 dpim

    NA

    ~40 to 1000 fold change compared to mock

    [136]

    VEEV

    V3526

    IA/B Trinidad donkey

    BALB/c, 6 to 8 week oldC3H/HeN mice, 6 to 8 week old

    105 PFU

    s.c.

    Single dose

    105 PFU of TrD, 28 dpim

    NP

    ND

    [137][138][139][140]

    Cynomolgus macaques (age not specified)

    2.5 × 106 PFU

    s.c.

    Single dose

    ∼108 PFU VEEV IE 68U201, 8 wpim

    aerosol

    PRNT80, 28 to 2560

    Rhesus macaques (2 to 4 years old)

    1.3 × 105 or 7.5 × 104 PFU

    s.c. or i.t./i.s.

    Single dose

    ND

    ND

    PRNT80, ~80 to 300

    Human clinical trial, Phase 1

    25 or 125 PFU

    s.c.

    Single dose

    NA

    NA

    NA

    VEEV

    V4020

    IA/B Trinidad donkey

    BALB/c mice, 4 to 8 week old

    104 PFU

    s.c.

    Single dose

    104 PFU of VEEV TrD, 28 dpim

    s.c.

    PRNT80, 160 to1280

    [141][142]

    Cynomolgus macaques (age not specified)

    ~104 PFU

    s.c. in the right leg

    Single dose (or second dose at 2 x 104 PFU i.m. if did not seroconvert)

    106 to 107 PFU of the VEEV TrD, 73 dpim

    aerosol

    PRNT80, >640

    EEEV

    5′U4&6 C65-69 E71-77 3′U11337 mutants

    FL93-939

    CD-1 mice, 5 to 6 week old

    1.5 × 105 GE

    s.c. in footpad, or i.c.

    Single dose

    105 PFU EEEV FL93, 21 dpim

    s.c. in both footpads

    PRNT80, 16 to ~4000

    [143]

    Live-attenuated (IRES)

                     

    CHIKV

    CHIKV/IRES

    LR2006 OPY1

    A129 mice, 3 or 10 week old

    104 PFU

    i.d.

    Single dose

    100 PFU LR2006 OPY1, 94 dpim

    i.d.

    PRNT80, >320

    [144][145]

    C57BL/6 mice, 3 week old

    105 PFU

    s.c. in the hind leg

    Single dose

    106.5 PFU Ross CHIKV, 21 dpim

    i.n.

    Mean PRNT80, 62

    A129 mice, 8 to 10 week old

    105 TCID50

    s.c.

    Single dose

    100 PFU LR2006 OPY1, 50 dpim

    i.d.

    Mean PRNT80, 1152

    Cynomolgus macaques, >3 years old

    105 PFU

    s.c. or i.d.

    Single dose

    105 PFU LR2006 OPY1, 52 dpim

    s.c. in the upper deltoid

    PRNT80, 40 to 640PRNT50, 160 to 1280

    ONNV

    CHIKV/IRES

    LR2006 OPY1

    A129 mice, 6 to 7 week old

    104 PFU

    i.d.

    Single dose

    105 PFU ONNV SG650, 38 dpim

    i.d.

    PRNT80, 160

    [146]

    VEEV

    ZPC/IRESv1, ZPC/IRESv2

    ID ZPC738

    CD-1 mice, 6 to 8 week old

    105 PFU

    s.c. in the scruff of the back

    Single dose

    105 PFU VEEV 3908, 4 wpim

    s.c. or aerosol

    PRNT80, 40 to 324

    [114]

    Cynomologous macaques, age not specified

    105 PFU

    s.c. in the upper deltoid

    Single dose

    ~ 8 × 105 to 9 × 106 PFU VEEV 3908, 35 dpim

    aerosol

    PRNT80, <20 to 20PRNT50, <20 to 160

    EEEV

    EEE/IRES

    FL93-939

    NIH Swiss mice, 3 to 4 week old

    104 PFU

    s.c. in the medial thigh

    Single dose

    103 PFU of FL93-939, 4 wpim

    i.p.

    PRNT80, 160 to 640

    [147]

    VEEV

    68U201/IRESv1 68U201/IRESv2

    IE 68U201

    CD1 mice, 6 to 8 week old

    105 PFU

    s.c. in right hind leg

    Single dose

    (Lethal dose, NP) 68U201 at 1, 3, or 12 mpim

    s.c.

    PRNT80, 64 to ~300

    [148][149]

    Cynomolgus macaques (age not specified)

    105 PFU

    s.c. in the upper deltoid

    Single dose

    4 × 104 PFU VEEV IE 68U201, 49 dpim

    aerosol

    PRNT80, ~100 to 340

    VEEV

    VEEV/IRES/C

    IA/B Trinidad donkey

    CD-1 mice, 8 week old

    105 PFU

    s.c.

    Single dose

    104 PFU of VEEV 3908, 6 wpim

    s.c.

    Mean PRNT80, 184

    [150]

    MAYV

    MAYV/IRES

    MAYV-CH

    BALB/c, 6 week old

    2 × 105 PFU

    s.c. i.pl. route

    Single dose

    2 × 105 PFU of WT MAYV, 28 dpim

    s.c. i.pl. route

    PRNT50, >640 (at 21dpi)

    [146][151][152]

    AG129

    2 × 104, 2 × 103 or 2 × 102 PFU

    s.c. i.pl. route

    Single dose

    2 × 103 PFU of WT MAYV, 14 dpim

    s.c. i.pl. route

    ND

    CD-1, 28-day old

    105 PFU

    s.c. over the dorsum

    Single dose

    ND

    ND

    PRNT80, 160 to ≥ 640

    AG129, 5 to 8 week old

    104 PFU

    i.d. on the left foot

    Single dose

    104 PFU of WT MAYV, 29 dpim

    s.c.

    PRNT80, 320 to ≥ 640

    Inactivated

                     

    CHIKV

    Vero cell adapted

    DRDE-06

    Swiss albino mice, 3 to 4 week old

    10, 25 or 50 ug

    s.c.

    Three doses (0, 14 and 28 days)

    ND

    ND

    PRNT90, 6400

    [153]

    CHIKV

    BPL/formalin-inactivated CHIKV

    BBV87 (in clinical trials)

    IND-06-AP3

    BALB/c mice, 4 to 6 week old

    10, 20 or 50 μg

    i.m.

    Two doses (0 and 14 days)

    2.5 x 104 TCID50 IND-06-AP3, 4 or 22 wpim

    i.n.

    GMT, NT50, 80 to 1280

    [154]

    Human clinical trial, Phase 1

    10, 20 or 30 μg

    i.m.

    Three doses (0, 29 and 57 days)

    NA

    NA

    NA

    [155]

    RRV

    Vero cell culture-derived whole-virus RRV vaccine Ross River Virus (RRV) Vaccine

    T48

    CD-1 mice, 7 to 8 week old

    0.0025, 0.01, 0.039, 0.156, 0.625, 2.5 or 10 μg

    s.c.

    Two doses (0 and 28 days)

    106 TCID50 RRV T48, 42 dpim

    i.v.

    Mean NT, ≤2.9 to 46.2

    [73][115][128][129]

    A129 mice, 7 to 8 week old

    0.063, 0.25 or 1 μg

    i.m.

    Two doses (0 and 21 days)

    102.5 TCID50 T48, 42 dpim

    s.c. into left footpad

    Mean NT, ≤14 to 21

    CD-1 mice, age not specified

    10 μg

    s.c.

    Two doses (0 and 28 days)

    106 TCID50 T48, 6 wpim

    i.v.

    1000 TCID50

    Guinea pigs (Duncan Hartley), age not specified

    10 μg

    s.c.

    Single or two doses (0 and 6 weeks)

    106 TCID50 T48, 10 or 34 wpim

    i.v.

    NP

    Human clinical trial, Phase 1/2

    1.25, 2.5, 5, or 10 μg

    i.m.

    Three doses in escalation (0, 21 days, 6 months)

    NA

    NA

    GMT, 50 to 520.9

    Human clinical trial, Phase 3

    2.5 ug

    i.m.

    Three doses (0, 3 weeks, 6 months)

    NA

    NA

    μNT GMT, ~0 to 85

    EEEV

    TSI-GSD-104 (formalin inactivated)

    PE-6

    Human clinical trial, Phase 2

    NP

    s.c. (0 and 28 days), i.d. (6 months)

    Three doses (0, 28 days and 6 months)

    NA

    NA

    PRNT80 >40 in 60% subjects (primary doses) versus 84% subjects (completed the 2-dose primary series and the 6-month dose)

    [156][157][158]

    EEEV

    fCVEV1219

    iCVEV1219

    gCVEV1219

    CVEV1219

    BALB/c mice, 6 to 8 week old

    0.1 to 5 µg of inactivated EEEV

    i.n., s.c. or i.m.

    Single dose or two doses (0 and 28 days)

    Lethal dose of EEEV FL93-939, at 28 dpim (single dose) or 56 dpim (two doses)

    aerosol

    PRNT80, ~1 to 1000

    [159]

    VEEV

    V3526 virus

    V3526

    BALB/c mice, 6 week old

    0.2 μg (s.c.) or 0.04 μg (i.m.)

    s.c. or i.m.

    Two doses (0 and 28 days)

    104 PFU VEEV TrD, 56 dpim

    aerosol or s.c.

    GMT PRNT80, ~60 to 2500

    [160]

    VEEV

    F-iV3526

    V3526

    BALB/c mice, 8 to 10 weeks old

    1, 3 or 5 μg

    i.n., s.c. (under the skin over the neck) or i.m. (thigh muscle of the hind leg)

    Single dose

    454 (i.n.), 897 (i.m.) or 55 (s.c.) PFU VEEV-TrD, 56 dpim

    aerosol

    Microneutralization titer of 100 to 3500

    [161][162]

    Virus-like particle

                     

    CHIKV

    VRC 311

    Or

    VRC-CHKVLP059-00-VP/ PXVX0317 (in clinical trials)

    37997

    BALB/c mice, 6 to 8 week old

    19 μg

    i.m.

    2 doses (2 and 5 weeks)

    ND

    ND

    IC50, 10703 to 54600

    [116][117][118][163]

    Cynomolgus macaques, 3 to 4 years old

    20 μg

    i.m.

    3 doses (0, 4 and 24 weeks)

    1010 PFU LR2006 OPY1, 15 wpim

    i.v.

    IC50, 10219 to 15072

    Human clinical trial, Phase 1

    10, 20 or 40 μg

    i.m.

    3 doses (0, 4 and 24 weeks)

    NA

    NA

    IC50, 4525 to 8745

    Human, clinical trial Phase 2

    20 μg

    i.m.

    2 doses (0 and 28 days)

    NA

    NA

    EC50 GMT, 2005

    Human clinical trial (Phase 2b, recruitment completed)

    6, 10 or 20 μg

    NP

    Two doses (0 and 14 or 28 days)

    NA

    NA

    NA

    CHIKV

    Baculovirus-expressed VLP

    S27

    AG129, 6 week old

    1 μg

    s.c.

    2 doses (0 and 21 days)

    1000 TCID50 S27, 6 wpim

    i.p.

    PRNT95, 40 to 80

    [164][165]

    C57BL/6 mice, 6 to 12 week old

    0.1 or 1 μg

    s.c.

    Single dose

    104 CCID50 LR2006 OPY1, 6 wpim

    s.c.

    NT95, ~1,100

     

    CHIKV

    Yeast-expressed VLP

    DRDE06/DRDE07

    BALB/c mice, 4 week or 2 days old

    10, 20 or 40 ug

    s.c.

    Three doses (0, 14 and 28 days)

    ND

    ND

    NT50, 128 to 2048

    [166]

    VEEV

    Venezuelan Equine Encephalitis Monovalent Virus-Like Particle Vaccine (VEEV)

    NA

    Human clinical trial (Phase 1, not recruiting)

    2, 10, or 20 μg

    i.m.

    Dose escalation (0, 28 days, and day 140 booster)

    NA

    NA

    NA

    [167]

    WEEV, EEEV, and VEEV

    VRC-WEVVLP073-00-VP (Trivalent vaccine)

    WEEV CBA87, EEEV PE-6 and VEEV TC-83

    BALB/c mice, 6 to 8 week old

    monovalent (5 μg) or trivalent (5 μg each)

    i.m.

    Two doses (0 and 21 days)

    2.5 × 103 PFU WEEV CBA87, 8.9 × 103 PFU EEEV FL93-939, and 1.3 × 103 PFU VEEV Trinidad donkey, 56 dpim

    aerosol

    PRNT80, ~250 to 100000

    [168]

    Cynomolgus macaques, age not specified

    Monovalent (20 μg) or trivalent (20 μg each)

    i.m.

    Two doses (0 and 28 days)

    106 PFU WEEV CBA87, 108 PFU EEEV FL93-939, and 108 VEEV Trinidad donkey, 56 dpim

    aerosol

    PRNT80, ~1000 to 10000

    Human clinical trial, Phase 1

    6, 30 or 60 μg

    i.m.

    Dose escalation (0 and 8 weeks)

    NA

    NA

    NA

    [169]

    DNA/RNA

                       

    VEEV

    VEEV 26S DNA plasmid

    I/AB TrD

    BALB/c mice, 6 to 8 week old

    ∼3 μg

    DNA/gene gun, delivered to two sites on the abdomen of each mouse

    Three doses (at 3-week intervals)

    ∼104 PFU of TrD, 9 wpim

    s.c., aerosol

    PRNT50, GMT <1.6 to 2.5

    [170][171]

    Hartley guinea pigs, age not specified

    ~5 μg

    DNA/gene gun, delivered to two sites on the abdomen of each mouse

    Three doses (0, 4 and 8 weeks)

    ∼104 PFU of TrD, 21 wpim

    aerosol

    PRNT50, 0 to 640

    VEEV

    DNA-Ad

    TC-83

    BALB/c mice, 6 to 8 week old

    1 μg of DNA per dose and 107 PFU of RAd/VEEV #3 per boost

    gene guni.n.

    immunised with the DNA vaccines on day 0, 14 and 28 and Ad-based vaccine on day 42

    100 LD50 of virulent airborne VEEV, 63 dpim

    aerosol

    PRNT50, 160

    [172]

    VEEV

    AG4-1C7

    AG4-1G2 AG2-5A7

    AG2-5A10 plasmid DNA

    I/AB TrD

    BALB/c mice, 6 to 8 week old

    4 μg

    particle-mediated epidermal delivery (i.d.)

    Three doses (at 3-week intervals)

    ∼104 PFU of VEEV TrD (≥1000 LD50), 70 dpim

    aerosol

    PRNT80, ~1 to 5.5 log10 GMT

    [173]

    VEEV

    pTC83 iDNA

    TC-83

    BALB/c mice, 3 week old

    50 μg

    i.m. electroporation

    Single dose

    105 PFU VEEV 3908, 21 dpim

    s.c.

    PRNT80, 10 to 320

    [174]

    WEEV

    pE3-E2-6K-E1

    pE3-E2

    P6K-E1

    71V-1658

    BALB/c, age not specified

    2 μg

    gene gun

    Three doses (14 days apart)

    1500 PFU WEEV 71V-1658,

    Fleming, or CBA87, 42 dpim

    i.n.

    ND

    [175]

    CHIKV

    pCHIKV-Capsid, pCHIKV-Envelope (pMCE321)

    Consensus

    C57BL/6 mice, 3 to 4 week old

    25 µg, 2–3 times

    Electroporation

    Two doses (2 weeks apart)

    ND

    ND

    ND

    [176][177][178]

    C57BL/6 mice, 6 to 8 week old

    25 μg

    i.m. electroporation

    Three doses (0, 14 and 21 days)

    7log10 PFU of PC-08, 35 dpim

    i.n.

    NP

    BALB/c mice

    25 μg

    i.m. electroporation

    Two doses (2 weeks apart)

    7log10 PFU PC-08

    i.n.

    TCID50, 20 to 320

    Rhesus macaques, age not specified

    1 mg

    i.m. electroporation

    Three doses (4 weeks apart)

    ND

    ND

    TCID50, 80 to 1280

    CHIKV

    Δ5nsP3 and Δ6K DNA

    LR2006 OPY1

    C57BL/6 mice, 5 to 6 week old

    20 μg

    i.d. with DermaVax electroporation

    Single dose or two doses (0 and 3 weeks)

    106 PFU LR2006 OPY1, 7 wpim

    s.c.

    NT50, 100 to 10000

    [131]

    CHIKV

    CHIKV-NoLS RNA

    LR2006 OPY1

    C57BL/6 mice, 28 days of age

    2 μg

    s.c. in the ventral/lateral side of the right foot

    Single dose

    104 PFU LR2006 OPY1, 30 dpim

    s.c. in the ventral/lateral side of the right (ipsilateral) or left (contralateral)

    PRNT80, 0

    [126]

    AG129 mice, 28 days old

    2 μg

    s.c. in the ventral/lateral side of the right foot

    Single dose

    104 PFU LR2006 OPY1, 30 dpim

    s.c. in the ventral/lateral side of the right (ipsilateral) or left (contralateral)

    ND

    VEEV, WEEV and EEEV

    3-EEV

    VEEV IAB TrD, WEEV CBA874 and EEEV FL91-46794

    C57BL/6 mice, 6 to 8 week old

    15 μg

    i.m. electroporation

    Two doses (0 and 21 days)

    104 PFU VEEV IAB TrD or 2 × 104 PFU WEEV CBA874 or 105 PFU EEEV FL91-46794, 7 wpim

    aerosol

    PRNT80, ~1 to 1000

    [179]

    MAYV

    scMAYV-E

    NA

    C57BL/6 mice, 5 to 8 week old

    25 μg

    i.m. electroporation

    Single, two doses or three doses (at 2 week intervals)

    ND

    ND

    PRNT50, 789.8

    [180]

    A129 mice, 4 to 6 week old

    25 μg

    i.m. electroporation

    Single, two doses or three doses (at 2 week intervals)

    102 PFU MAYV 15537

    i.p.

    ND

    CHIKV

    p181/25-7

    TSI-GSD-28

    BALB/c mice, 3 week old

    10 μg

    i.m. electroporation

    Single dose

    6 × 106 PFU CHIKV Ross, 28 dpim

    i.n.

    PRNT80, 160 to 1280

    [181]

    CHIKV

    dMaB

    NA

    BALB/c mice, age not specified

    100 μg

    Electroporation

    Single dose

    107 PFU Del-03

    s.c. or i.n.

    IC50, 3 to 4.5log10

    [182]

    CHIKV

    iRNAΔ5nsP3

    iDNAΔ5nsP3

    LR2006 OPY1

    C57BL/6 mice, 8 week old

    0.125, 1.25 or 10 μg

    i.m. in the gastrocnemius muscle of the left hind leg

    Single dose

    106 PFU LR2006 OPY1, 5 wpim

    s.c. at the dorsal side of each hind foot

    NT50, ~1 to 104

    [183]

    VEEV

    pMG4020 DNA plasmid

    TC-83

    BALB/c, 4 to 8 week old

    0.5 or 5 ug

    i.m. electroporation

    Single dose

    104 PFU VEEV TrD, 28 dpim

    s.c.

    PRNT80, 320 to >1280

    [141]

    VEEV

    VEEVWT VEEVCOCAP

    VEEVCO

    IAB TrD

    BALB/c, 6 to 8 week old

    25, 5, or 1 μg

    i.m. electroporation

    Two doses (3 weeks apart)

    ∼104 PFU VEEV IAB strain TrD, 7 wpim

    aerosol

    PRNT80, 1 to ~4.5log10

    [184][185]

    New Zealand White rabbits, age not specified

    500 μg of VEEVCO

    i.m. electroporation

    Three doses (0, 28 and 230 days)

    ND

    ND

    PRNT80, ~3log10 to 5log10

    Cynomolgus macaques, age not specified

    50 or 500 μg of VEEVCO

    i.m. electroporation

    Two doses (0 and 56 days)

    3 × 108 PFU VEEV IAB TrD

    aerosol

    PRNT80, ~0.8log10 to 3.5log10

    Human clinical trial, Phase 1

    0.5 or 2 mg

    i.m. electroporation or i.d. electroporation

    Three doses (days 0, 28, and 56)

    NA

    NA

    GMT PRNT80, 7 to 78

    WEEV

    pVHX-671V-1658

    pVHX-6 CBA87

    pVHX-6 Fleming

    Fleming, CBA 87 or 71V-1658,

    BALB/c mice, age not specified

    2 shots × 2.5 μg precipitated on 0.5 mg gold

    gene gun

    Four doses (2 weeks apart)

    1.5 × 103 PFU WEEV Fleming, CBA 87 or 71V-1658, 8 wpim

    i.n.

    ND

    [186]

    WEEV and EEEV

    LANAC E1ecto

    WEEV McMillan

    CD-1 mice, 4 to 6 week old

    10 μg

    s.c. injection dorsal to the cervical spine

    Two doses (2 weeks apart)

    104 PFU WEEV McMillan, Montana-64, or EEEV Florida-93, 4, 5, 9, 11, or 13 wpim

    i.n. or s.c.

    PRNT50, <40 to 200

    [187]

    CHIKV

    mRNA-1388 (or VAL-181388 in clinical trials)

    NA

    Human clinical trial, Phase 1

    25, 50 or 100 μg

    i.m.

    Dose escalation procedure (0 and 4 weeks)

    ND

    ND

    ‘dose-dependent increase’ in neutralizing and binding antibody titers

    [188]

    CHIKV

    mRNA-1944

    SL15649

    AG129, age not specified

    0.4, 1 or 10 mg/kg

    i.v. tail vein injection

    Single dose

    102.5 TCID50 of CHK

    subcutaneous injection in the footpad and hock of the right leg

    ND

    [189][190]

    Cynomolgus macaques, 2 to 3 year old

    0.5 mg/kg

    i.v.

    Single dose

    ND

    ND

    FRNT50, 5 to 12

    Human clinical trial, Phase 1 (active, not recruiting)

    0.1, 0.3 and 0.6 mg/kg

    i.v.

    Dose escalation

    NA

    NA

    NT50, ‘all participants also showed circulating neutralizing antibody activity’

    Subunit

                       

    CHIKV

    CHIKV-sE1 and -sE2

    S27

    AG129 mice, 6 week old

    2 μg

    s.c.

    Two doses (0 and 21 days)

    1000 TCID50 of S27 isolate, 9 wpim

    i.p.

    NT95, <25

    [164][165][191]

    CHIKV

    rE2p

    IND-06-AP3

    BALB/c, 6 to 8 week old

    10, 20 or 50 μg

    i.m.

    Two doses (2 weeks apart)

    Mice immunized with 50 μg challenged with 7 log10 TCID50 /mL, 4 or 22 wpim

    i.n.

    NT50, 0.25log10 to 2.5log10

    [154]

    CHIKV

    CHIKE1 and CHIKE2 recombinant proteins

    DRDE-06

    BALB/c

    40 μg

    s.c.

    Three doses (0, 21 and 35 days)

    ND

    ND

    PRNT90, 32 to 512

    [192]

    Chimeric virus

                     

    Measles virus-based chimeras

                 

    CHIKV (VLP)

    MV-CHIKV

    06–49

    CD46-IFNAR, 6 week old

    103 to 105 TCID50

    i.p.

    Single or two doses (30 days apart)

    100 PFU of CHIKV 06-49, 2 mpim

    i.p.

    PRNT50, 450 to 4050

    PRNT90, 50 to 450

    [193][194][195][196][197]

    Cynomolgus macaques, age not specified

    5 × 105 (± 0.5 log) TCID50

    i.m.

    Two doses (28 days apart)

    1.4 × 105 PFU LR2006 OPY1, 56 dpim

    s.c.

    PRNT80, 40 to >640

    Human clinical trial, Phase 1

    1.5 × 104, 7.5 × 104 or 3.0 × 105 TCID50

    i.m. or s.c.

    Dose escalation (0 and 28 days, or 0 and 90 days)

    NA

    NA

    PRNT50, 5 to 433

    Human clinical trial, Phase 2

    5 × 104 or 5 × 105 TCID50

    i.m.

    Three doses (0, 28, and 196 days)

    NA

    NA

    PRNT50, ~5 to 5000

    Alphavirus-based chimeras

                 

    CHIKV

    VEE/CHIKV

    EEE/CHIKV

    SIN/CHIKV

    LR2006 OPY1

    NIH Swiss, C57BL/6, >3 week old

    5.8 log10 PFU (VEE/CHIKV and SIN/CHIKV), 5.3 log10 PFU (EEE/CHIKV)

    s.c. in the medial thigh

    Single dose

    6.5 log10 PFU (Ross CHIKV strain), 21 dpim

    i.n.

    PRNT80, 20 to 320

    [198]

    CHIKV

    VEE/IRES-CHIKV

    VEE/IRES-C/CHIKV

    NA

    A129 mice, 6 to 9 week old

    104 PFU

    s.c.

    Single dose

    102 PFU of LR2006 OPY1, 5 weeks post immunization

    s.c.

    PRNT80, >640

    [199]

    CHIKV

    EILV-CHIKV

    CHIKV 996659

    C57BL/6 mice, 4 week old

    8.8 log10 PFU

    s.c.

    Single dose

    6 log10 PFU 99659, 30 dpim

    i.d.

    PRNT80, ≥ 80

    [125][220]

    IFNα/βR−/−, 6 week old

    8.8 log10 PFU

    s.c.

    Single dose

    3 log10 PFU 99659, 292 dpim

    i.d.

    PRNT80, 160 to 1280

    Cynomolgus macaques, 3 to 5 years

    8.1 log10 PFU

    i.m. into the right quadriceps

    Single dose

    5 log10 PFU LR2006 OPY1, 31 dpim

    s.c.

    PRNT80, 80 to 640

    EEEV

    EILV/EEEV

    EEEV FL-93

    Adult CD-1 mice (age not specified)

    108 PFU

    s.c.

    Single dose

    105 PFU EEEV-FL93, 70 dpim

    i.p.

    PRNT80, 80 to 640

    [125][220]

    EEEV

    Trivalent EILV/EEEV EILV/VEEV EILV/CHIKV

    EEEV FL-93, VEEV IAB TC-83, CHIKV 996659

    Adult CD-1 mice (age not specified)

    108 PFU

    s.c.

    Single dose

    105 PFU EEEV-FL93, 70 dpim

    i.p.

    PRNT80, 40 to 640 and 20 to 640 for mono- and trivalent vaccines respectively

    VEEV

    EILV/EEEV

    VEEV IAB TC-83

    Adult CD-1 mice (age not specified)

    108 PFU

    s.c.

    Single dose

    103 PFU VEEV-IC 3908, 70 dpim

    s.c.

    PRNT80, 80 to 1280

    VEEV

    Trivalent EILV/EEEV, EILV/VEEV EILV/CHIKV

    EEEV FL-93, VEEV IAB TC-83, CHIKV 996659

    Adult CD-1 mice (age not specified)

    108 PFU

    s.c.

    Single dose

    103 PFU VEEV-IC 3908, 70 dpim

    s.c.

    PRNT80, 40 to 640 and 20 to 80 for mono- and trivalent vaccines respectively

    EEEV (Sindbis virus)

    SIN/NAEEEV

    EEEV FL93-939

    NIH Swiss mice, 8 week old

    3.7, 4.7 or 5.7 log10 PFU

    s.c.

    Single dose

    6 log10 PFU FL93-939, 28 dpim

    i.p.

    PRNT80, 125 to 660

    [200]

    SIN/SAEEEV

    EEEV BeAr436087

    NIH Swiss mice, 8 week old

    3.8, 4.8 or 5.8 log10 PFU

    s.c.

    Single dose

    6 log10 PFU FL93-939, 28 dpim

    i.p.

    PRNT80, 28 to 308

    VEEV

    SIN-83

    VEEV IAB TC-83

    Weanling NIH Swiss mice, 6 day old

    103, 104, 105 or 106 PFU

    s.c.

    Single dose

    106 PFU VEEV IC ZPC738 IC SH3

    s.c.in medial thigh

    PRNT80, 30 to 960

    [201][202]

    NIH Swiss mice, 6 week old

    5 × 105 PFU

    s.c.

    Two doses

    2 x 105 or 106 PFU VEEV ZPC738, 8 wpim

    s.c., i.c., or i.n.

    PRNT80, 55 to 73 (single), 100 to 160 (booster)

    SAAR/TRD

    VEEV IAB TrD

    NIH Swiss mice, 6 week old

    5 × 105 PFU

    s.c.

    Two doses

    2 x 105 or 106 PFU VEEV ZPC738, 8 wpim

    s.c., i.c., or i.n.

    PRNT80, 126 to 167 (single), 152 to 160 (booster)

    SIN/TRD

    VEEV IAB TrD

    NIH Swiss mice, 6 week old

    5 × 105 PFU

    s.c.

    Two doses

    2 x 105 or 106 PFU VEEV ZPC738, 8 wpim

    s.c., i.c., or i.n.

    PRNT80, 37 to 57 (single), 50 to 73 (booster)

    SIN/ZPC

    VEEV ID ZPC738

    NIH Swiss mice, 6 week old

    5 × 105 PFU

    s.c.

    Two doses

    2 x 105 or 106 PFU VEEV ZPC738, 8 wpim

    s.c., i.c., or i.n.

    PRNT80, 187 to 253 (single), 253 to 487 (booster)

    All the above

    VEEV IAB TC-83, IAB TrD, ID ZPC738

    Syrian golden hamsters, 6 week old

    5 × 105 PFU

    s.c. in the medial thigh

    Single dose

    106 PFU

    s.c.in medial thigh

    ND

    WEEV

    SIN/CO92

    WEEV CO92-1356

    NIH Swiss mice, 6 week old

    3.5, 4.5, or 5.0 log10 PFU

    s.c. in the medial thigh

    Single dose

    5.3 log10 PFU WEEV TBT235, 28 dpim

    i.n.

    PRNT80, 20 to 640

    [203]

    SIN/SIN/McM

    WEEV McMillan

    NIH Swiss mice, 6 week old

    4.8 or 5.8 log10 PFU

    s.c. in the medial thigh

    Single dose

    5.0 log10 PFU WEEV McMillan, 28 dpim

    i.n.

    PRNT80, 600 to 604

    SIN/EEE/McM

    EEEV 436087 and WEEV McMillan

    NIH Swiss mice, 6 week old

    4.6 or 5.6 log10 PFU

    s.c. in the medial thigh

    Single dose

    5.0 log10 PFU WEEV McMillan, 28 dpim

    i.n.

    PRNT80, 416 to 420

    Vaccinia virus-based chimeras

                 

    CHIKV

    MVA-CHIKV

    LR2006-OPY1

    C57BL/6 mice, 6 to 8 week old

    107 PFU (first dose), 2 × 107 PFU (second dose)

    i.p.

    Two doses (2 weeks apart)

    106 PFU LR2006-OPY1, 9 wpim

    s.c. in the dorsal side of each hind foot

    NT50, ~100 to 3000

    [204]

    CHIKV

    MVA-CHIK

    LR2006-OPY1

    BALB/c mice, 4 to 6 week old

    107 TCID50 units

    i.d. injection into the left hind footpad.

    Single or two doses (28 days apart)

    104

    LR2006 OPY1 TCID50 units at 39 or 42 dpim

    i.d.

    TCID50, 5 to 15

    [205]

    AG129, 6 to 10 week old

    107 TCID50 units

    i.d. injection into the left hind footpad.

    Single or two doses (28 days apart)

    102

    LR2006 OPY1 TCID50 units at 39 or 42 dpim

    i.d.

    TCID50, 4 to 8

    CHIKV

    MVA-6KE1, MVA-E3E2, MVA-6KE1E3E2

    CHIKV S27

    AG129 mice, 7 week old

    5 × 106 TCID50

    i.m. into the quadriceps muscles of the left leg

    Two doses (3 weeks apart)

    103

    TCID50 CHIKV-S27 and CHIKV-IND/NL10, 63 dpim

    i.p.

    NT100, 10 to 160

    [206]

    EEEV, VEEV, and WEEV

    MVA-BN-E/V/W (monovalent)

    MVA-BN-E + MVA-BN-V + MVA-BN-W (triple mixture of monovalent vaccines)

    MVA-BN-WEV (trivalent)

    WEEV 71 V-1658, EEEV FL93-939NA and VEEV TrD

    BALB/c mice, age not specified

    108 TCID50

    s.c. or i.m.

    Two doses (28 days apart)

    5 × 103 or 104 PFU of WEEV Fleming, EEEV PE6, or VEEV TrD, 14 days post booster

    i.n.

    NT50, ~750 to 3800 (monovalent), ~<60 to 340 (triple mixture of monovalent vaccines) and ~<60 to 380 (trivalent)

    [207]

    Adenovirus-based chimeras

                 

    CHIKV

    CAdVax-CHIK

    LR2006 OPY1

    CD-1 or C57BL/6, 6 to 8 week old

    108 IU

    i.p.

    Single dose

    104 CCID50 LR2006 OPY1 or QIMR, 6.5 wpim

    s.c. into side of each hind foot towards the ankle

    NT100, ~2000

    [208]

    CHIKV

    ChAdOx1 Chik

    NA

    BALB/c, 6 to 8 week old

    108 IU

    i.m.

    Single dose

    ND

    ND

    NT50, 5.39 × 103

    [209][210]

     

    AG129, 5 week old

    108 IU

    i.m. in each leg

    Single dose

    9.7 × 104 PFU LR2006 OPY1, 30 dpim

    i.d. into the left foot

    ND

     

    ChAdOx1 Chik

    ChAdOx1 Chik ΔCap

    AG129, 5 week old

    108 IU

    i.m. in each hind leg

    Single dose

    9.7 × 104 PFU of LR2006 OPY1, 30 dpim

    i.d. into the left foot towards the ankle

    PRNT80, 32 to 64 (Chik), 16 to 32 (Chik ΔCap)

    [211]

     

    CHIK001 (in clinical trials)

    Human clinical trial, Phase 1

    5 × 109, 2.5 × 1010 or 5 × 1010 vp

    i.m.

    Single dose

    ND

    ND

    ND

    [212]

    MAYV

    ChAdOx1 May

    NA

    AG129, 5 week old

    1.6 × 104 PFU

    i.m. in each leg

    Single dose

    1.6 × 104 PFU MAYV-CH, 30 dpim

    i.d. into the left foot

    PRNT50, 160 to 620

    [210]

    VEEV

    Rad/VEEV#3

    VEEV IAB TC-83

    BALB/c, 6 to 8 week old

    107 PFU

    i.n.

    Three doses (at 0, 7 and 21 days)

    Dose ND, 28 dpim

    aerosol

    PRNT50 (NP)

    [213]

    BALB/c, 6 to 8 week old

    107 PFU

    i.n.

    Two doses (at 0, 21 days)

    5000 LD50 TrD, 42 dpim

    aerosol

    ND

    [214]

    WEEV

    Ad5-WEEV

    WEEV 71V-1658

    BALB/c mice, age not specified

    107 PFU

    i.m.

    Single or two doses (at 4 weeks)

    1.5 × 103 PFU Fleming or 71V-1658, 13 wpim

    i.n.

    PRNT50, 160

    [215]

    WEEV

    Ad5-E1

    WEEV 71V-1658

    BALB/c mice, 6 to 9 week old

    107 PFU

    i.m. in both leg

    Single dose

    50 LD50 of 71V-1658, 7 dpim, or

    400 LD50 CBA87, 1, 3, 5 or 7 dpim

    i.n.

    PRNT50, <10

    [216]

    Vesiculovirus-based chimeras

                 

    CHIKV

    rVSVΔG-CHIKV

    CHIKV S27

    C57BL/6, 3 week old

    106 PFU

    i.m. into the right hind leg muscle

    Single dose

    104 PFU LR 2006 OPY1, 30 dpim

    s.c. in the left rear footpad

    PRNT80, 80 to 640

    [217]

    VEEV

    rVSIV-VEEV

    VEEV ZPC738

    CD-1, 4 to 6 week old

    108/107 PFU

    i.m.

    Single dose

    104 PFU VEEV ZPC738, 35 or 245 dpim

    s.c.

    PRNT80, 288 to 600 at 25 and 35 dpim, 304 to 360 at 245 dpim

    [218]

    VEEV

    rISFV-VEEV

    VEEV ZPC738

    CD-1, 4 to 6 week old

    108 PFU

    i.m.

    Single dose

    104 PFU VEEV ZPC738, 28 dpim

    s.c.

    PRNT80, ≥20

    CD-1, 4 to 6 week old

    108 PFU

    i.m.

    Single dose

    104 PFU VEEV ZPC738, 35 or 245 dpim

    s.c.

    PRNT80, 40 to 160 at 25 and 35 dpim, 25 to 64 at 245 dpim

    EEEV

    rISFV-EEEV

    EEEV FL93-939

    CD-1, 4 to 6 week old

    108 PFU

    i.m.

    Single dose

    104 PFU EEEV FL93-939, 28 dpim

    s.c.

    PRNT80, ≥20

    Epitope-based

                     

    CHIKV

    E2EP3

    NA

    C57BL/6 mice, 3 week old

    100 μg (50 μg for booster doses)

    s.c. in the abdominal flank

    Three doses (0, 14 and 21 days)

    106 PFU CHIKV SGP11, 30 dpim

    s.c. region at the ventral side of the right hind footpad, towards the ankle

    ~40% reduction from mock control

    [23]

    1 s.c., subcutaneous; i.v., intravenous; i.m., intramuscular; i.d., intradermal; i.p., intraperitoneal; i.n., intranasal; i.t./i.s., intrathalamic/ intraspinal; i.pl., intraplantar; i.c., intracranial; dpim, days post immunization; wpim, weeks post immunization; mpim, months post immunization; IRES, internal ribosome entry site; PFU, plaque forming units; TCID50, 50% tissue culture infective dose; CCID50, 50% cell culture infectious dose; IC50, 50% inhibitory concentration; GE, genomic equivalents; IU, infectious units; AID50, 50% animal infectious dose; PRNT50, 50% plaque reduction neutralizing antibody titer; PRNT80, 80% plaque reduction neutralizing antibody titer; PRNT90, 90% plaque reduction neutralizing antibody titer; LD50, median lethal dose; NT50, 50% neutralizing titer; GMT, geometric mean titer; μNT, neutralizing titer; SIN, Sindbis virus; ISFV, Isfahan virus; May, Mayaro virus; EILV, Eilat virus, VSV/VSIV, vesicular stomatitis virus; MV, measles virus; MVA, modified vaccinia virus Ankara; NP, not provided; NA, not applicable; WT, wild type. Data curated from literature reported through February 2021.

    The entry is from 10.3390/microorganisms9050899

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