The alphavirus E3 glycoprotein is a multifunctional component of the process of synthesis and maturation of the viral surface glycoprotein spikes. In addition to acting as a signal to initiate translocation of viral glycoproteins across the membranes of the rough endoplasmic reticulum, E3 also promotes the heterodimerization and intracellular processing of E1 and PE2 glycoproteins (23
). Furin cleavage of PE2 into E2 and E3 activates the spikes to a metastable state capable of initiating infection (52
). By analogy with the prM protein of dengue Flavivirus
), furin cleavage of PE2 may begin after transit of an acidic late component of the Golgi body, where E3 is thought to suppress the acid pH-triggered activation of glycoprotein E1 fusion capability. Although the pr fragment produced by furin cleavage of prM of dengue is released after return to neutral pH, in the case of alphaviruses E3 may be released (2
) or retained on the mature virus particles (32
Although elimination of the furin cleavage site from PE2 in VEEV is a lethal mutation (8
), pseudorevertant viruses were rescued after transfection of transcripts of cleavage deletion genomic cDNA clones due to second-site suppressor mutations in the E1, E2, or E3 glycoprotein genes (17
). The stability of the cleavage deletion mutation (24
) and attenuated phenotype of the virus prompted the evaluation of V3526 as a vaccine for animals and humans (34
). In the course of preclinical evaluation of V3526 as a live vaccine, a series of MAbs were generated to further characterize the immune response elicited by vaccination with V3526.
The MAbs described in this report bound a previously unrecognized epitope on the E3 glycoprotein. Reconstructions of a cleavage-site deletion mutant of Sindbis virus revealed that the E3 portion of the PE2 molecule in the virus forms a prominent lobe on the distal portion of the glycoprotein spike (32
). The potent neutralization of V3526 infectivity by MAb 13D4 was likely due to interference with cell surface binding or penetration as previously reported for the E2c
-specific MAbs (38
The furin cleavage of alphavirus PE2 glycoprotein begins in the trans
-Golgi network (29
). The binding of MAb 13D4 to wild-type virus-infected cells indicates that E3 is present on the cell surface, but we were unable to determine whether the target is E3 or residual PE2. The observation that furin cycles between the Golgi body and the cell surface (30
) provides for the possibility that some processing occurs after PE2 appears on the plasma membrane. If true, this suggests several possible mechanisms by which anti-E3 antibodies may inhibit virus production. First, MAb 13D4 bound to PE2 might interfere with furin cleavage (10
). However, the incorporation of PE2 into nascent Alphavirus
spikes does not prevent the budding of particles (8
), and the qPCR analysis indicates that 13D4 reduces particle formation to very low levels, as shown in Table . Alternatively, antibody binding of PE2 on the cell surface may inhibit formation of the glycoprotein spike matrix and coalescence of the envelope required for viral budding. Finally, if MAb 13D4 is bound to E3 after its cleavage from PE2, the potent inhibition of wild-type virus production by 13D4 would suggest the existence of an undescribed role for E3 in a critical late event preceding release of the virion from the cell. Efforts are ongoing to identify the nature of the E3 entity present on the cell surface.
Protection against lethal Alphavirus
challenge by passive immunization with antibodies that do not inhibit plaque formation was demonstrated previously (41
). The ability of the six E3-specific MAbs described here to inhibit plaque formation by V3526 virus is not directly relevant to protection against wild-type viral challenge. However, it does correspond to the level of protection afforded by passive immunization. The expanded repertoire of antibody isotypes detected after challenge indicates that a significant level of viral replication occurs in passively immunized mice. These observations indicate anti-E3 MAb likely protects animals against wild-type VEEV by inhibiting the amplification of the challenge virus, subsequently moderating viremia and the progression of disease until the adaptive immune response is capable of resolving the infection. In contrast, the E2c
MAb provided apparent sterile immunity when passively transferred to mice, based on the lack of de novo
antibody responses following challenge.
This study is the first in which the E3 protein has been demonstrated to contain protective epitopes. These findings are directly relevant to ongoing efforts toward developing licensable equine encephalitis virus vaccines and therapeutics. TC-83 is a live VEEV vaccine currently available for limited use in humans. The vaccine has a variable reactogenicity rate but also fails to induce a detectable serum neutralizing antibody response in ca. 20% of recipients. Because a neutralizing antibody response is currently accepted as the surrogate marker of protection against laboratory infection by VEEV, TC-83 nonresponders receive subsequent vaccinations with C-84 vaccine, an inactivated vaccine also available for use in at-risk laboratory investigators. However, C-84 is prepared by formalin inactivation of TC-83 and therefore, is unlikely to contain detectable E3 glycoprotein. Although the lack of E3 is unlikely to be responsible for the failure rate, an inactivated vaccine developed from a cleavage mutant virus, such as V3526, might be expected to elicit a broadened antibody response and provide an additional level of protection due to antibody to the E3 epitope. In addition, development of inactivated vaccines from cleavage-site mutants of WEEV and EEEV may likewise increase their effectiveness, since we found that an anti-E3 MAb specific for the WEEV E3 glycoprotein also affords protection in mice (unpublished observation). In addition to vaccine considerations, MAbs to this protective epitope on the E3 glycoprotein could be a promising component of an antibody-based therapeutic for alphaviruses.