Selection of a Rare Neutralization-Resistant Variant following Passive Transfer of Convalescent Immune Plasma in Equine Infectious Anemia Virus-Challenged SCID Horses

Vaccines preventing HIV-1 infection will likely elicit antibodies that neutralize diverse strains. However, the capacity for lentiviruses to escape broadly neutralizing antibodies (NAbs) is not completely understood, nor is it known whether NAbs alone can control heterologous infection. Here, we determined that convalescent immune plasma from a horse persistently infected with equine infectious anemia virus (EIAV) neutralized homologous virus and several envelope variants containing heterologous principal neutralizing domains (PND). Plasma was infused into young horses (foals) affected with severe combined immunodeficiency (SCID), followed by challenge with a homologous EIAV stock. Treated SCID foals were protected against clinical disease, with complete prevention of infection occurring in one foal. In three SCID foals, a novel neutralization-resistant variant arose that was found to preexist at a low frequency in the challenge inoculum. In contrast, SCID foals infused with nonimmune plasma developed acute disease associated with high levels of the predominant challenge virus. Following transfer to an immunocompetent horse, the neutralization-resistant variant induced a single febrile episode and was subsequently controlled in the absence of type-specific NAb. Long-term control was associated with the presence of cytotoxic T lymphocytes (CTL). Our results demonstrate that immune plasma with neutralizing activity against heterologous PND variants can prevent lentivirus infection and clinical disease in the complete absence of T cells. Importantly, however, rare neutralization-resistant envelope variants can replicate in vivo under relatively broad selection pressure, highlighting the need for protective lentivirus vaccines to elicit NAb responses with increased breadth and potency and/or CTL that target conserved epitopes.Development of an effective vaccine will be critical in the efforts to control the human immunodeficiency virus type 1 (HIV-1) pandemic. Unfortunately, vaccines evaluated in completed human efficacy trials have shown moderate to no protective effects, and, clearly, much more work is needed to define the correlates of lentivirus immune protection. Although these correlates are still not entirely known, vaccine strategies that elicit antibodies with broad neutralizing activity are currently of considerable interest, and it is widely believed that HIV-1 envelope glycoproteins that induce broadly neutralizing antibodies (NAbs) will be critical components of a protective vaccine (21, 28, 53, 63).Equine infectious anemia virus (EIAV) is a macrophage-tropic lentivirus that causes persistent infection in horses worldwide and serves as an important large-animal translational model in which to dissect basic correlates of protective lentiviral immunity (9, 31, 33, 38, 57). EIAV is a naturally occurring lentivirus, and infection results in a predictable course of recurrent episodes of plasma viremia and clinical disease. As with HIV-1 and simian immunodeficiency virus (SIV), EIAV infection is not cleared. However, infected horses eventually control viral replication and clinical disease to remain persistently infected inapparent carriers. Adaptive immune responses, including NAbs, are required for EIAV control since young horses (foals) with severe combined immunodeficiency (SCID), unlike normal foals, fail to eliminate the initial viremia following challenge (46). Equine SCID is caused by a frameshift mutation in the gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) (55, 60) and has an autosomal recessive mode of inheritance (47). The equine SCID defect is more severe than its murine counterpart in that SCID foals are incapable of forming either coding or signal joints (55). Adoptive transfer of EIAV-specific T and B lymphocytes to a SCID foal results in functional cytotoxic T lymphocytes (CTL) and NAb activity and is protective against homologous EIAV challenge (33).During acute EIAV infection, each recurrent episode coincides with the emergence of an antigenically distinct EIAV variant as defined by type-specific NAb, which neutralizes virus isolated during early disease episodes but not virus isolated during subsequent disease episodes (2, 20, 22, 43, 52). Amino acid variation primarily occurs within hypervariable regions V1 to V8 of the envelope gp90 surface unit (SU) and particularly within the V3/principal neutralizing domain (PND) region (1, 19, 24, 25, 57). Our work with EIAV-infected SCID foals indicates that significant envelope diversification does not occur in the absence of NAbs but that rapid envelope diversification occurs when adaptive immune responses are reconstituted (35). Thus, adaptive immunity, including NAb, drives selection of EIAV envelope variants during acute infection. Amino acid changes occur primarily within the V3 to V7 hypervariable SU regions, and many changes affect potential N-linked glycosylation sites (PNLGS) (35). Importantly, however, CTL also target the SU, and variants that escape CTL recognizing an EIAV V3/PND epitope have been identified (37, 38). Thus, both NAbs and CTL are capable of contributing to the selection of EIAV SU variants, but the relative contributions of each to such selection are not known.Recently, SU variation was evaluated in an immunocompetent pony experimentally inoculated with the virulent wild-type Wyoming strain of EIAV (57). Seventy-one distinct V3 variants that partitioned into five major nonoverlapping groups were identified and designated PND1 to PND5. Neutralization assays using chimeric infectious molecular clones containing these PNDs suggested a transition from type-specific NAb responses toward more broadly reactive immune responses during the course of infection and indicated that genetic changes conferring resistance to broadly NAbs lead to recrudescence of clinical disease following a lengthy clinically quiescent period (57). Thus, the NAb response broadens significantly during long-term persistent EIAV infection, and broadly NAbs play a critical role in EIAV immune control.Studies of nonhuman primates have provided important information regarding the protective effects of NAbs. Passive immunization of macaques with purified immunoglobulin from chimpanzees infected with several different HIV-1 isolates results in complete protection from homologous chimeric simian/human immunodeficiency virus (SHIV) infection when the immunoglobulin is given 24 h prior to challenge (54). Passive transfer of a triple combination of broadly neutralizing human monoclonal antibodies directed against the envelope of a primary HIV-1 isolate results in complete protection against SHIV infection in some macaques while others become infected but exhibit decreased plasma viremia (29). The contribution of T cells to partial protection in these studies is not clear, and the presence or absence of viral escape variants in the unprotected macaques has not been evaluated. In neonatal macaques, various combinations of broadly neutralizing human monoclonal antibodies directed against conserved HIV envelope epitopes administered before and after SHIV challenge result in protection against persistent systemic infection in some animals, but clinical disease develops in others (12-14). Virus-specific T-cell proliferative responses are detected in some of the protected animals, indicating that cellular immune responses occur and likely contribute to protection by eliminating infected cells (13).Despite the fact that NAbs can block experimental SHIV infection, selection pressure exerted by NAbs plays a critical role in HIV-1 and SIV envelope evolution during infection, and evasion of NAb responses is an important mechanism of HIV-1 and SIV persistence (11, 16, 27, 48, 59). The maturation of a type-specific NAb response in SIV-infected rhesus macaques significantly correlates with diversification in the V1/V2 region of the SIV envelope (50). In HIV-1, NAbs are detectable within the first 2 months postinfection and result in an early and significant selection force on the virus population (49). Escape from NAbs involves many amino acid substitutions with little cross-neutralization between closely related strains, and NAb responses drive the diversification of the HIV-1 envelope during the early stages of infection (16). The early appearance of NAbs in patients with acute HIV-1 infection results in the replacement of neutralization-sensitive virus by successive populations of resistant virus, and virus escape primarily involves changes in N-linked glycosylation (59). Thus, overcoming neutralization escape constitutes a significant barrier to the ultimate efficacy of any NAb-eliciting HIV-1 vaccine.Because the SCID defect occurs naturally in the horse, it provides a powerful and unique opportunity to finely dissect the protective effects of immune interventions against a naturally occurring lentivirus independent of other de novo adaptive immune responses. This level of dissection is not possible in other lentivirus model systems. The goal of the current study was to determine if broadly NAbs could protect against lentivirus challenge in the complete absence of T lymphocytes and other adaptive immune responses. We hypothesized that convalescent immune plasma from a long-term persistently infected inapparent carrier horse containing antibodies capable of neutralizing homologous and several heterologous EIAV SU PND variants would provide complete protection when infused into SCID foals before experimental virus inoculation. This plasma was administered to four SCID foals 24 h prior to challenge, and four control SCID foals received normal horse plasma. Clinical outcome, plasma viral load, and serum neutralization activity were analyzed in all foals. Although complete protection was achieved in one treated foal, infection occurred in the others. In foals that became viremic, the SU sequence and neutralization phenotype of the breakthrough virus were determined. As part of these experiments, blood containing this virus was inoculated into a naive immunocompetent horse, and the adaptive immune responses associated with its control were further evaluated.