Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses

Abstract

Background: Chikungunya virus (CHIKV) and other alphaviruses are the etiologic agents of numerous diseases in both humans and animals. Despite this, the viral mediators of protective immunity against alphaviruses are poorly understood, highlighted by the lack of a licensed human vaccine for any member of this virus genus. The alphavirus E2, the receptor-binding envelope protein, is considered to be the predominant target of the protective host immune response. Although envelope protein domains have been studied for vaccine and neutralization in flaviviruses, their role in alphaviruses is less characterized. Here, we describe the role of the alphavirus E2 domains in neutralization and protection through the use of chimeric viruses.

Methodology/principal findings: Four chimeric viruses were constructed in which individual E2 domains of CHIKV were replaced with the corresponding domain from Semliki Forest virus (SFV) (ΔDomA/ΔDomB/ΔDomC/ ΔDomA+B). Vaccination studies in mice (both live and inactivated virus) revealed that domain B was the primary determinant of neutralization. Neutralization studies with CHIKV immune serum from humans were consistent with mouse studies, as ΔDomB was poorly neutralized.

Conclusions/significance: Using chimeric viruses, it was determined that the alphavirus E2 domain B was the critical target of neutralizing antibodies in both mice and humans. Therefore, chimeric viruses may have more relevance for vaccine discovery than peptide-based approaches, which only detect linear epitopes. This study provides new insight into the role of alphavirus E2 domains on neutralization determinants and may be useful for the design of novel therapeutic technologies.

Fig 1. Genome organization of chimeric CHIKV/SFV viruses.

Genome organization of chimeric CHIKV/SFV viruses. The different domains of E2 from SFV were inserted into the CHIKV genome in the corresponding position in individual constructs using a PCR based cloning approach. Each virus expressed the GFP protein under control of a second sub-genomic promoter. Red portions of the E2 represent genetic sequences of SFV whereas CHIKV is shown in blue.

Fig 2. Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV.

Adult C57bl/6 mice (n = 6) were infected with 10⁵ PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10⁵ PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.

Fig 3. Representative histopathology of spleen and brain of live-virus vaccinated C57bl/6 mice post-challenge.

Mice previously infected with 10⁵ PFU of CHIK or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) were challenged with 10⁵ PFU of SFV. Surviving mice were euthanized and spleens and brains were harvested followed by processing for Hematoxylin and Eosin (H&E) staining. A-F. Spleens at 10x magnification. The scale bars represent 100 μM. G-L. Hippocampal neurons at 25x magnification. The scale bar represents 50 μM. Arrows signify neuron degeneration.

Fig 4. Neutralization and protection following vaccination with UV-inactivated viruses.

Six-week old C57bl/6 mice were vaccinated with 5μg of each parental (CHIK or SFV) or chimeric viruses (ΔDomA, ΔDomB, ΔDomC, ΔDomA+B) inactivated with ultraviolet (UV) radiation. Mice received a second injection 28 days later. Groups of mice were then challenged with 10⁵ PFU of either CHIKV or SFV. A) Neutralizing antibody responses against both CHIK and SFV were assessed prior to challenge using a luciferase based assay. Infectious virus was mixed with a 1:20 dilution of serum and used to infect cells following incubation overnight at 4°C. Luciferase activity was measured in cell lysates after five hours of infection. Data are expressed as fold neutralization, normalized to mock serum. Mice challenged with SFV were monitored for B) weight loss and C) survival. Weight loss is expressed as the mean percentage of starting weight of the group. D) Change in footpad width was used as a marker of CHIKV disease. Data are presented as percent change as compared to pre-challenge levels.

Fig 5. Protection afforded by UV-inactivated parental or chimeric viruses.

Groups of mice previously vaccinated with 5μg prime and boost of each UV-inactivated parental (CHIKV or SFV) or chimeric (ΔDomA, ΔDomB or ΔDomC, ΔDomA+B) virus were challenged with 10⁵ PFU of either wild-type CHIKV or SFV. Five days post-infection, 3 mice from each group were euthanized and brains and footpads were harvested for SFV and CHIKV challenge groups, respectively. Tissues were processed for hematoxylin and eosin (H&E) staining. A-G. Hippocampal neurons from SFV challenged mice using a 25x objective. (A) Uninfected control, (B) CHIKV, (C) SFV, (D) ΔDomA, (E) ΔDomB, (F) ΔDomC, (G) ΔDomA+B. Scale bars represent 50 μM. Black arrows signify neuron degeneration. H-N. Decalcified footpads of CHIKV challenged mice using a 10x objective. (H) Uninfected control, (I) CHIKV, (J) SFV, (K) ΔDomA, (L) ΔDomB, (M) ΔDomC, (N) ΔDomA+B. Scale bars represent 100 μM. White arrows represent areas of mononuclear cell infiltration into the tissue.

Fig 6. Neutralization of parental and chimeric viruses with CHIKV-immune human serum.

Convalescent serum samples from 10 human patients were collected aseptically. Neutralization was performed using plaque reduction neutralization 50 (PRNT50) test. Two-fold dilutions of serum samples were incubated individually with each parental (CHIKV or SFV) or chimeric virus (ΔDomA, ΔDomB or ΔDomC, ΔDomA+B). Cells were fixed 36 hours post-infection and stained with crystal violet solution to visualize plaques. Data is presented as the inverse of the final serum dilution that showed 50% reduction or greater in plaques.