Production and Characterization of SIV Envelope-Specific Rhesus Monoclonal Antibodies from a Macaque Asymptomatically Infected with a Live SIV Vaccine

Abstract

Five rhesus monoclonal antibodies (RhMAbs) were produced by rhesus EBV transformation of peripheral blood B cells from a rhesus macaque that had been asymptomatically infected with an attenuated, macrophage-tropic SIV strain, 17E-Cl. These MAbs recognized conformation-dependent epitopes on SIV gp120 and could not be mapped using synthetic peptides. All five RhMAbs were able to neutralize the vaccine strain and a heterologous isolate, SIV/DeltaB670. The RhMAbs did not cross-react with HIV-2; by contrast, four human MAbs derived from an HIV-2-infected person were broadly cross-reactive with both SIV and HIV-2 gp120s. Cross-competition analysis indicated that the five RhMAbs could be placed in two groups recognizing two nonoverlapping epitopes; while the HMAbs were placed in two additional competition groups. Binding of the three group I RhMAbs (1.7F, 3.11B, and 1.10A) as well as HMAb 17A was shown to be sensitive to specific amino acid alterations in V4 occurring in natural env variants. The results of this study demonstrate that RhEBV transformation provides a means to probe rhesus antibody responses to SIV infection at the monoclonal level. RhMAbs will allow structural and functional studies of envelope glycoprotein determinants that elicit protective immune responses against SIV.

Dissecting the Humoral Immune Response to Simian Immunodeficiency Virus: Mechanisms of Antibody-Mediated Virus Neutralization

“…Similar antibody maturation profiles have been observed in other lentiviral systems including equine infectious anemia virus (EIAV) (20), simian/human immunodeficiency virus (SHIV) (21), and human immunodeficiency virus (HIV) (21,22). Based on this observed maturation of the immune system associated with protective immunity, we isolated monoclonal antibodies (MAbs) from protected monkeys in an effort to further characterize the protective SIV-specific antibody response (23,24).…”

“…We generated a unique panel of MAbs derived from the peripheral blood of four rhesus macaques infected with attenuated SIV/17E-CL for more than 8 mo, the time-point at which the envelope-specific polyclonal antibody response had achieved maturation and the monkeys were protected from a pathogenic virus challenge (6,16,23,24). Thus, these MAbs are reflective to some degree of the protective immune response generated to attenuated SIV infection.…”

“…It has been established that one common mechanism of neutralization exhibited by envelope-specific antibodies is the ability to block the primary receptor, CD4 (27)(28)(29)(30)(31)(32). While several antibodies capable of inhibiting CD4 binding have been derived from immunizing mice for relatively short periods of time with recombinant SIV envelope proteins (33,34), none of the rhesus MAbs generated to date have blocked SIV gp120-CD4 binding by ELISA (23,24). Because the MAbs do not block CD4 binding yet manage to neutralize virus, we performed assays to determine if neutralizing MAbs inhibited fusion.…”

“…The panel of rhesus MAbs described above bound with similar efficiency to envelope proteins from various strains of SIV in a number of solid phase assays, including (i) viral envelope proteins expressed on cells infected with several strains of SIV (including SIV/ 17E-CL, SIVmac239, SIVmac251 and SIVdeltaB670) (23,24), (ii) SIV/17E-CL soluble gp140 trimers produced in a mammalian system in a ConA ELISA, and (iii) soluble gp120 monomers produced in a baculovirus expression system (SIVmac251) or viral gp120 (SIV/ 17E-CL) in nonreducing Western blot analyses. Using a selected panel of MAbs representing each of the competition groups, we performed real-time binding studies using surface plasmon resonance (SPR) as measured on a Biacore 3000 to determine the kinetics of MAb binding to recombinant, trimeric SIV gp140 proteins (26).…”

Kinetic Rates of Antibody Binding Correlate with Neutralization Sensitivity of Variant Simian Immunodeficiency Virus Strains

“…We have developed a panel of rhesus MAbs derived from monkeys infected with attenuated SIV/17E-CL that identify at least eight binding domains on gp120 (9,45). This panel of MAbs represents both linear and conformational binding domains, including three groups of neutralizing MAbs that recognize discontinuous epitopes in the C-terminal half of SIV gp120.…”