Chikungunya Virus Neutralization Antigens and Direct Cell-to-Cell Transmission Are Revealed by Human Antibody-Escape Mutants

Abstract

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop “groove” as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.

Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

“…Antibody production and purification. Procedures for producing MAbs 8B10 and 5F10 have been previously reported (15,16). Fab frag- ments were purified from the IgG after papain digestion using a protein A column and then further purified over a Superdex 200 16/600 column.…”

“…The CHIKV-neutralizing antibodies 8B10 and 5F10 had been previously isolated from CD-40-activated B cells of infected patients (15) and have been shown to protect mice from CHIKV infection (16). However, CHIKV with a V216E or R82G mutation on E2 were also able to propagate in the presence of antibody 5F10 (16).…”

“…Similarly, CHIKV with mutations T12I or R82G on domain A of E2 or T101M on domain II of E1 (16) were able to propagate in the presence of antibody 8B10. When mutant CHIKV containing these escape mutations to antibody 8B10 were propagated in the presence of 8B10, the virus particles migrated to cellular junctions (15), indicative of direct cell-to-cell transmission. Thus, antibody 8B10 enhances the direct transmission of mutant CHIKV between cells.…”

Viperin restricts chikungunya virus replication and pathology

“…qRT-PCR reaction mixes were done in 12.5 μl reaction volume and performed in Applied Biosystems (ABI) 7900HT Fast Real-Time PCR System in 384-well plates, with the following conditions: (a) reverse transcription step (50°C for 30 minutes; 1 cycle); (b) PCR initial activation step (95°C for 15 minutes; 1 cycle); (c) 2-step cycling (94°C for 15 seconds, follow by 60°C for 1 minute; 45 cycles). Viral load was estimated from a standard curve generated using serial dilution of synthetic CHIKV negative-strand nsP1 RNA transcripts as described previously (66).…”

Serological correlates of immune protection conferred by Chikungunya virus infection

“…The cytokines Il-1, Il-6, and Il-10 have been identified as pro-inflammatory markers in the acute phase, and mCP-1, Il-6, Il-8, mIP-1α, mIP-1β, and Th-1 activation with viral persistence in macrophages were identified as markers in the chronic phase of the disease with severe clinical symptoms (2,8,9). In population studies of ChIKV disease, antibody titers have been estimated by IgG and Igm antibody elISA (2,10,11) and neutralizing antibodies by the hemagglutination-inhibition test (hIt), complement-fixation test and by serum microneutralization (SNt) tests (12,13). The 50% plaque reduction neutralization test (PRNt 50 ) was used in clinical evaluation of ChIKV vaccine in a Phase II study (14).…”