Mechanisms of Equine Infectious Anemia Virus Escape from Neutralizing Antibody Responses Define Epitope Specificity

Abstract

Determining mechanisms of viral escape to particular epitopes recognized by virus-neutralizing antibody can facilitate characterization of host-neutralizing antibody responses as type- versus group-specific, and provides necessary information for vaccine development. Our study reveals that a single N-glycan located in the 5′ region of the Wyoming wild-type equine infectious anemia virus (EIAV) principal neutralizing domain (PND) accounts for the differences in neutralization phenotype observed between PND variants, while variations in charged amino acids within the PND do not appear to play a key role in viral escape. Site-directed mutagenesis and peptide mapping of a conserved epitope to neutralizing antibody in the 3′ region of the PND showed rapid selective pressure for acquisition of a 5′ PND N-glycan responsible for defining the specificity of the neutralizing-antibody response.

Summary

D etermining mechanisms of viral escape to particular epitopes recognized by virus-neutralizing antibody can facilitate characterization of host-neutralizing antibody responses as type- versus group-specific, and provide necessary information for vaccine development. Our study reveals that a single N-glycan located in the 5′ region of the Wyoming wild-type equine infectious anemia virus (EIAV) principal neutralizing domain (PND) accounts for differences in neutralization phenotype observed between PND variants, while variation in charged amino acids within the PND do not appear to play a key role in viral escape. Site-directed mutagenesis and peptide mapping of a conserved epitope to neutralizing antibody in the 3′ region of the PND showed rapid selective pressure for acquisition of a 5′ PND N-glycan responsible for defining the specificity of the neutralizing-antibody response.

EIAV is a macrophage-tropic lentivirus of horses that results in lifelong infection (23). The clinical form of disease is characterized by viremia, fever, thrombocytopenia, and signs of malaise, which are interspersed with periods of clinical quiescence and low viremia. In 1973, Kono et al. proposed antigenic variation as a model of lentiviral persistence for EIAV (16), suggesting that recurrent febrile episodes are due to replication of antigenic variant viruses that escape from neutralizing antibody (16). Evidence from diverse lentiviruses has demonstrated that glycosylation can alter the immunogenicity and antigenicity of the surface glycoprotein (3), and that asparagine-linked glycans (N-glycans) play a key role in conferring neutralization escape, probably through induction of conformational changes of the envelope glycoprotein, and/or by masking epitopes for neutralizing antibody (6,17,26)

Sponseller et al. previously described the genetic and phenotypic changes in EIAV associated with escape from type-specific antibody during the acute-to-chronic transition of EIAV (24). In that study, Pony 524 was experimentally infected with the Wyoming wild-type inoculum (EIAV_Wyo), and developed a characteristic clinical disease course of recurring episodes of fever interspersed with afebrile periods ranging from days to months. The predominant genotypes of the EIAV PND were identified throughout infection, including the inapparent stage of disease, and were the basis for the molecular clones used to study immune selection of EIAV envelope variants throughout infection, including the inapparent stage of disease. However, the genotype that was predominant in the inoculum and during acute disease temporally decreased with development of an apparent variant-specific, autologous, neutralizing-antibody response. Escape from the variant-specific neutralizing antibody response was accompanied by four amino acid changes, three of which were within the EIAV V3-containing PND. Two of the amino acid changes in the PND encoded a putative N-glycan (PNG) motif.

Here we report the molecular basis of escape from type-specific antibody in a horse (Pony 524) experimentally infected with EIAV_Wyo, demonstrating the specific role of N-glycans and charge in the EIAV V3 in mediating escape from variant-specific virus-neutralizing antibody. In addition, we identify the specific V3-located epitope that was recognized by autologous (Pony 524) sera, and that was associated with neutralization of virus containing wild-type PND from the inoculum. Further, we demonstrate that heterologous virus resistant to neutralization by the same antibody response effective in neutralizing EIAV_PND1 could be rendered susceptible by deletion of the N-glycan corresponding to the one absent in the wild-type inoculum.

Others have demonstrated that net charge of the lentiviral envelope can influence the biological phenotypes of cell tropism, fusogenicity, and resistance to neutralization (5,7 –9,13,15,22, 27). To determine if charge is important in mediating escape of EIAV_PND1 from type-specific neutralizing antibody, we performed site-directed mutagenesis (12), and directionally cloned into pSPeiav19 (21), to generate a full-length infectious chimeric proviral clone (24). Using this strategy we mutated a proline to histidine (P199H), the amino acid site that differed in charge between EIAV_PND1 and EIAV_PND2 (Fig. 1), and performed a focal reduction immunoassay on provirus (performed in triplicate and repeated twice) (4), with autologous serum (from 89 days post-infection [dpi]) that neutralized EIAV_PND1, but not EIAV_PND2 (Fig. 1B). Significant differences in this immunoassay are ≥2-fold serial dilutions. The proline-to-histidine mutation, which changes an amino acid with a nonpolar side chain (proline) to one with a charged, polar side chain (histidine), did not result in change of neutralization titer between EIAV_PND1 and EIAV_PND1D, and therefore did not appear to contribute to the phenotypic differences in neutralization between EIAV_PND1 and EIAV_PND2 (Table 1). These results suggested that the PNG in EIAV_PND2 accounted for escape from antibodies that neutralized EIAV_PND1.

FIG. 1.

(A) Alignment and amino acid sequence identity of the chimeric infectious clones derived through site-directed mutagenesis. The EIAV V3 containing the PND is single underlined; V4 is double underlined. The location of the D_NT neutralizing epitope identified by Ball et al. (1) is underlined in bold. Putative N-glycans are highlighted in gray. The peptide mimetic sequence is in bold type. (B) Comparison of neutralization titers among EIAV_PND1 and EIAV_PND2 chimeras and parental backbone, SPeiav19, by autologous sera collected at sequential days post-infection (DPI). The virus titer is defined by a 75% reduction or greater in virus infectivity.

Table 1.

Neutralization of Chimeric Virus and Mapping of Neutralization Epitope

Serum	EIAV_PND1	EIAV_PND2	EIAV_PND1C	EIAV_PND1D	SPeiav19	SPeiav19D
Autologous^a	128^b	<4	<8	64	<4	256
Autologous+peptide mimetic-1^c	16	ND^e	ND	ND	<4	<8
Autologous+peptide mimetic-HIV^d	128	ND	ND	ND	ND	256

Autologous serum from 89 days post-infection.

Neutralization titers are the reciprocal of the highest serum dilution resulting in a 75% reduction in focus-forming units with differences for this assay at or greater than two dilutions.

Peptide mimetic-1 (VNESTEYWGFKWLEC) incubated with virus and autologous serum.

Peptide mimetic-HIV control (PIDNASYRLISCNTS) incubated with virus and autologous serum.

Not done.

N-glycans are characterized by asparagine-glycosidic attachments where an N-acetylglucosamine is bound to the amide nitrogen of asparagine associated with the N-X-S/T amino acid motif. Differences between EIAV_PND1 and EIAV_PND2 include a D184N substitution paired with an N186T substitution, consistent with gain of a putative N-linked glycosylation site. In addition to this 5′ variable PNG site, three other conserved PNG sites are present, all of which might play a role in the masking of epitopes to neutralizing antibodies (26) (Fig. 1A). N-linked glycans may influence neutralization of virus through conformational changes of the surface glycoprotein, and/or masking of epitopes for neutralizing antibody (6,25). To determine whether N-glycan acquisition is important in mediating the escape of EIAV_PND1 from what appeared to be type-specific neutralizing antibody, the PNG present in EIAV_PND2, yet absent in EIAV_PND1, was added to EIAV_PND1 by site-directed mutagenesis, yielding EIAV_PND1C (Fig. 1A). Neutralization assays with EIAV_PND1C demonstrated that this PNG was sufficient for escape from the neutralizing antibody response that was effective in inhibiting replication of EIAV_PND1 (Table 1). Therefore, resistance to apparent EIAV_PND1-specific neutralizing antibody appeared to be causally associated with a single N-glycan located in the 5′ end of the PND-containing EIAV_PND1 V3. Previous studies by Howe et al. provide data from a panel of natural variant EIAV envelope isolates that support the V3 domain as the principal domain recognized by neutralizing antibody; however, in that study resistance to neutralization was cooperatively mediated by changes in glycosylation of V4 (14). Our studies are consistent with V3-containing epitopes to neutralizing antibody; however, the present study extends the understanding of N-glycan variation in EIAV V3, by demonstrating that changes in glycosylation in V3 alone can also mediate naturally-occurring escape from neutralizing antibody.

As SPeiav19 was the backbone for EIAV_PND1 (24), this infectious molecular clone with a heterologous V3 enabled determination of whether the 5′ putative N-glycan motif located in the SPeiav19 PND played a role in neutralization phenotype by masking a conserved epitope for neutralizing antibody. The genetic differences between SPeaiv19 and EIAV_PND1 are limited to the V3–V4 subregion of the surface glycoprotein, allowing differences in neutralization phenotype to be ascribed to this region. We previously demonstrated that the neutralizing antibody response to EIAV_PND1 differed from the response to SPeiav19, in both the time of onset and rate of development, indicating that EIAV_PND1 was antigenically distinct from SPeiav19. In addition, the longitudinal neutralizing antibody response to SPeiav19 was similar to that observed for the heterologous EIAV_WSU-5 isolate (2) (data not shown), and thus likely represents a broadly neutralizing antibody response (2,10,11). However, the virus-neutralizing antibody response to SPeiav19 was essentially the same as that to EIAV_PND2, leading us to conclude in previous studies that EIAV_PND2 and SPeiav19 were neutralized by an antibody response of similar specificity (24). The differences in specificity between EIAV_PND1 and EIAV_PND2, and the similar development of neutralizing antibodies to both EIAV_PND2 and SPeaiv19, together with sequence homology in the 3′ region of the EIAV-PND, suggested that the 3′ region of the PND contains an epitope that could be recognized by antibodies in Pony 524 89-dpi serum, if the SPeaiv19 N-glycan in the PND were removed.

To determine if the epitope were conserved in heterologous EIAV, yet potentially masked by a similarly located PNG in the 5′ region of the PND, we disrupted the NXS/T motif with an N186D substitution, yielding SPeiav19D. SPeiav19 contains a V3 very similar to the one used to map the EIAV PND epitopes to neutralizing antibody using murine monoclonal antibodies (1,18). Given the observation that epitope D_NT, previously identified by Ball et al. (1), shares sequence homology with the downstream region of EIAV_PND1, we speculated that serum derived 89 dpi from Pony 524 would be able to neutralize SPeiav19D (Fig. 1A). SPeaiv19D was susceptible to neutralization with antibody present in serum from Pony 524 89 dpi, suggesting not only that the 5′ putative N-glycan of SPeaiv19 is glycosylated, but that the epitope recognized by neutralizing antibody in Pony 524 89-dpi serum is in the 3′ homologous region of the EIAV V3, as the remaining viral sequence is the same (Fig. 1A). Interestingly, the 3′ region of the EIAV V3 loop is largely conserved in characterized North American EIAV isolates (18,24).

To demonstrate whether the epitope that was protected by the V3-located N-glycan that conferred escape from type-specific neutralizing antibody mapped to the EIAV V3, we used a sequence-specific peptide to EIAV_PND1 (VNESTEYWGFKWLEC), to adsorb serum-containing neutralizing antibody that recognized epitopes contained in this region of the PND (19,20). Admixture of homologous peptide sequences with serum otherwise effective in mediating neutralization of virus, resulted in virus replication with neutralization titers equivalent to negative controls (Table 1). Therefore, the EIAV PND contains an epitope recognized by autologous serum containing neutralizing antibodies from an EIAV-infected horse. These results also suggest that this epitope is linear, and is approximately located where the epitope D_NT was identified (1). Interestingly, the downstream region of EIAV_PND1, EIAV_PND2, SPeaiv19, and SPeaiv19D, share the amino acid sequence “EYWGFKWLEC,” suggesting that the epitope recognized by autologous serum is located within this motif; epitope D_NT also contains this amino acid sequence. Interestingly, the 3′ region of the EIAV V3 loop is largely conserved in North American EIAV isolates (18,24) suggesting that this downstream epitope is largely conserved, yet frequently masked by an N-glycan in the 5′ region of V3. Taken together, our results indicate that the masking of epitopes by N-glycans and other mechanisms of escape need to be considered when defining the specificity of epitopes to neutralizing-antibody responses.

Footnotes

Acknowledgments

We thank Susan Carpenter and Robert Mealey for helpful discussions.

Author Disclosure Statement

No conflicting financial interests exist.

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