Vaccine induced antibodies to the first variable loop of human immunodeficiency virus type 1 gp120, mediate antibody-dependent virus inhibition in macaques

Abstract

The role of antibodies directed against the hyper variable envelope region V1 of human immunodeficiency virus type 1 (HIV-1), has not been thoroughly studied. We show that a vaccine able to elicit strain-specific non-neutralizing antibodies to this region of gp120 is associated with control of highly pathogenic chimeric SHIV(89.6P) replication in rhesus macaques. The vaccinated animal that had the highest titers of antibodies to the amino terminus portion of V1, prior to challenge, had secondary antibody responses that mediated cell killing by antibody-dependent cellular cytotoxicity (ADCC), as early as 2 weeks after infection and inhibited viral replication by antibody-dependent cell-mediated virus inhibition (ADCVI), by 4 weeks after infection. There was a significant inverse correlation between virus level and binding antibody titers to the envelope protein, (R=-0.83, p=0.015), and ADCVI (R=-0.84 p=0.044). Genotyping of plasma virus demonstrated in vivo selection of three SHIV(89.6P) variants with changes in potential N-linked glycosylation sites in V1. We found a significant inverse correlation between virus levels and titers of antibodies that mediated ADCVI against all the identified V1 virus variants. A significant inverse correlation was also found between neutralizing antibody titers to SHIV(89.6) and virus levels (R=-0.72 p=0.0050). However, passive inoculation of purified immunoglobulin from animal M316, the macaque that best controlled virus, to a naïve macaque, resulted in a low serum neutralizing antibodies and low ADCVI activity that failed to protect from SHIV(89.6P) challenge. Collectively, while our data suggest that anti-envelope antibodies with neutralizing and non-neutralizing Fc(R-dependent activities may be important in the control of SHIV replication, they also demonstrate that low levels of these antibodies alone are not sufficient to protect from infection.

Figure 1. Relative efficacy of Tev vaccines in macaques

(A) The expression of Env, Nef, Rev, and Tev proteins was assessed by western blot in cell lysates at 4, 8, 14, and 18 hours post-transfection of the pHXB2 molecular clone in 293T cells. (B) Amino acid sequence alignment of the putative HIV_HXB2, HIV_Ba-L, HIV_SF162, and HIV_89.6 Tev proteins. (C) HIV_Ba-L, HIV_HXB2, HIV_SF162, and HIV-1_89.6 Tev proteins expressed in HEK 293 cells. The Tev proteins produced by the cDNAs were identified using antibodies to Rev. The 89.6 Tev protein was purified from E. coli using a mouse anti-HIV-1 Tat column and separated on 10-20 % SDS-PAGE (right panel). Tev protein was recognized by Rabbit antibody to Rev (lane 1), but not with normal rabbit serum (lane 2). Molecular weight marker is shown in lane 3. (D) Study design. The HIV_Ba-L, HIV_89.6, and HIV_SF162 Tev cDNAs were used in each DNA immunization, where “prot” stands for boosts with the purified HIV_89.6 Tev protein. Challenge exposure to SHIV_89.6P was performed via the intravenous route. (E) Plasma virus levels in vaccinated and control macaques (left and center panels) following challenge exposure to SHIV_89.6P. Right panel depicts mean level for both groups. (F) CD4⁺ T-cell counts in vaccinated and control macaques (left and center panels) following challenge exposure to SHIV_89.6P. Right panel depicts median level for both groups.

Figure 1. Relative efficacy of Tev vaccines in macaques

(A) The expression of Env, Nef, Rev, and Tev proteins was assessed by western blot in cell lysates at 4, 8, 14, and 18 hours post-transfection of the pHXB2 molecular clone in 293T cells. (B) Amino acid sequence alignment of the putative HIV_HXB2, HIV_Ba-L, HIV_SF162, and HIV_89.6 Tev proteins. (C) HIV_Ba-L, HIV_HXB2, HIV_SF162, and HIV-1_89.6 Tev proteins expressed in HEK 293 cells. The Tev proteins produced by the cDNAs were identified using antibodies to Rev. The 89.6 Tev protein was purified from E. coli using a mouse anti-HIV-1 Tat column and separated on 10-20 % SDS-PAGE (right panel). Tev protein was recognized by Rabbit antibody to Rev (lane 1), but not with normal rabbit serum (lane 2). Molecular weight marker is shown in lane 3. (D) Study design. The HIV_Ba-L, HIV_89.6, and HIV_SF162 Tev cDNAs were used in each DNA immunization, where “prot” stands for boosts with the purified HIV_89.6 Tev protein. Challenge exposure to SHIV_89.6P was performed via the intravenous route. (E) Plasma virus levels in vaccinated and control macaques (left and center panels) following challenge exposure to SHIV_89.6P. Right panel depicts mean level for both groups. (F) CD4⁺ T-cell counts in vaccinated and control macaques (left and center panels) following challenge exposure to SHIV_89.6P. Right panel depicts median level for both groups.

Figure 2. Immune responses in the vaccinated/challenged macaques

(A) ELISPOTs were performed using HIV_Ba-L, HIV_SF162, and HIV_89.6 Tev peptide pools for the stimulation of macaque blood. ELISPOT responses were also obtained using Concanavalin A, a stimulus to control for cell viability (data not shown). Antibody titers to Tev (B), Tat(C), Rev(D), and the pooled HIV_Ba-L, SHIV_SF162P3, and SHIV_89.6P Envelope proteins (E). The arrows refer to DNA immunizations (weeks 0, 4, 8), protein boosts (weeks 12, 18), and challenge exposure (week 22); titers to the purified Tev, Tat, Rev, and pooled HIV_Ba-L, HIV_SF162P3, and HIV_89.6P Envelope proteins. The weeks 0, 2, 6, and 10 refer to the weeks post challenge (p.c.). Data from control macaques are not shown. (F) Overlapping peptides of the entire HIV_89.6 Tev protein whose sequence is depicted in Fig. 1B were used with the sera (1:100 dilution) of the macaques after immunization (weeks 10, 19) and challenge exposure (weeks 2, 5 p.c.) in some of the vaccinated and control macaques. The area included in the dotted line area defines the V1 peptides (16 to 23), proceeded by the peptides derived from the first exon of Tat and followed by the peptides derived from the second exon of Rev. (G) Sera (1:100 dilution) of all immunized macaques after challenge exposure (weeks 2, 5 p.c.) and the two control macaques L915 and M320 were reacted with overlapping peptides 16–28 for the 89.6 V1. (H) Amino acid sequences of the 21-24 overlapping peptides recognized by the sera of macaques M308, M316, and M490 and M607 within the V1 region of SHIV_89.6P. (I): Sera (1:100 dilution) of the immunized macaques M218, M308, M316, and M490 before and after challenge exposure (weeks 10 and 19 and 2, 5 respectively) and the two control macaques. L915 and M320 were reacted with overlapping peptides 16–28 for the SF162 V1 region.

Figure 2. Immune responses in the vaccinated/challenged macaques

(A) ELISPOTs were performed using HIV_Ba-L, HIV_SF162, and HIV_89.6 Tev peptide pools for the stimulation of macaque blood. ELISPOT responses were also obtained using Concanavalin A, a stimulus to control for cell viability (data not shown). Antibody titers to Tev (B), Tat(C), Rev(D), and the pooled HIV_Ba-L, SHIV_SF162P3, and SHIV_89.6P Envelope proteins (E). The arrows refer to DNA immunizations (weeks 0, 4, 8), protein boosts (weeks 12, 18), and challenge exposure (week 22); titers to the purified Tev, Tat, Rev, and pooled HIV_Ba-L, HIV_SF162P3, and HIV_89.6P Envelope proteins. The weeks 0, 2, 6, and 10 refer to the weeks post challenge (p.c.). Data from control macaques are not shown. (F) Overlapping peptides of the entire HIV_89.6 Tev protein whose sequence is depicted in Fig. 1B were used with the sera (1:100 dilution) of the macaques after immunization (weeks 10, 19) and challenge exposure (weeks 2, 5 p.c.) in some of the vaccinated and control macaques. The area included in the dotted line area defines the V1 peptides (16 to 23), proceeded by the peptides derived from the first exon of Tat and followed by the peptides derived from the second exon of Rev. (G) Sera (1:100 dilution) of all immunized macaques after challenge exposure (weeks 2, 5 p.c.) and the two control macaques L915 and M320 were reacted with overlapping peptides 16–28 for the 89.6 V1. (H) Amino acid sequences of the 21-24 overlapping peptides recognized by the sera of macaques M308, M316, and M490 and M607 within the V1 region of SHIV_89.6P. (I): Sera (1:100 dilution) of the immunized macaques M218, M308, M316, and M490 before and after challenge exposure (weeks 10 and 19 and 2, 5 respectively) and the two control macaques. L915 and M320 were reacted with overlapping peptides 16–28 for the SF162 V1 region.

Figure 2. Immune responses in the vaccinated/challenged macaques

(A) ELISPOTs were performed using HIV_Ba-L, HIV_SF162, and HIV_89.6 Tev peptide pools for the stimulation of macaque blood. ELISPOT responses were also obtained using Concanavalin A, a stimulus to control for cell viability (data not shown). Antibody titers to Tev (B), Tat(C), Rev(D), and the pooled HIV_Ba-L, SHIV_SF162P3, and SHIV_89.6P Envelope proteins (E). The arrows refer to DNA immunizations (weeks 0, 4, 8), protein boosts (weeks 12, 18), and challenge exposure (week 22); titers to the purified Tev, Tat, Rev, and pooled HIV_Ba-L, HIV_SF162P3, and HIV_89.6P Envelope proteins. The weeks 0, 2, 6, and 10 refer to the weeks post challenge (p.c.). Data from control macaques are not shown. (F) Overlapping peptides of the entire HIV_89.6 Tev protein whose sequence is depicted in Fig. 1B were used with the sera (1:100 dilution) of the macaques after immunization (weeks 10, 19) and challenge exposure (weeks 2, 5 p.c.) in some of the vaccinated and control macaques. The area included in the dotted line area defines the V1 peptides (16 to 23), proceeded by the peptides derived from the first exon of Tat and followed by the peptides derived from the second exon of Rev. (G) Sera (1:100 dilution) of all immunized macaques after challenge exposure (weeks 2, 5 p.c.) and the two control macaques L915 and M320 were reacted with overlapping peptides 16–28 for the 89.6 V1. (H) Amino acid sequences of the 21-24 overlapping peptides recognized by the sera of macaques M308, M316, and M490 and M607 within the V1 region of SHIV_89.6P. (I): Sera (1:100 dilution) of the immunized macaques M218, M308, M316, and M490 before and after challenge exposure (weeks 10 and 19 and 2, 5 respectively) and the two control macaques. L915 and M320 were reacted with overlapping peptides 16–28 for the SF162 V1 region.

Figure 2. Immune responses in the vaccinated/challenged macaques

(A) ELISPOTs were performed using HIV_Ba-L, HIV_SF162, and HIV_89.6 Tev peptide pools for the stimulation of macaque blood. ELISPOT responses were also obtained using Concanavalin A, a stimulus to control for cell viability (data not shown). Antibody titers to Tev (B), Tat(C), Rev(D), and the pooled HIV_Ba-L, SHIV_SF162P3, and SHIV_89.6P Envelope proteins (E). The arrows refer to DNA immunizations (weeks 0, 4, 8), protein boosts (weeks 12, 18), and challenge exposure (week 22); titers to the purified Tev, Tat, Rev, and pooled HIV_Ba-L, HIV_SF162P3, and HIV_89.6P Envelope proteins. The weeks 0, 2, 6, and 10 refer to the weeks post challenge (p.c.). Data from control macaques are not shown. (F) Overlapping peptides of the entire HIV_89.6 Tev protein whose sequence is depicted in Fig. 1B were used with the sera (1:100 dilution) of the macaques after immunization (weeks 10, 19) and challenge exposure (weeks 2, 5 p.c.) in some of the vaccinated and control macaques. The area included in the dotted line area defines the V1 peptides (16 to 23), proceeded by the peptides derived from the first exon of Tat and followed by the peptides derived from the second exon of Rev. (G) Sera (1:100 dilution) of all immunized macaques after challenge exposure (weeks 2, 5 p.c.) and the two control macaques L915 and M320 were reacted with overlapping peptides 16–28 for the 89.6 V1. (H) Amino acid sequences of the 21-24 overlapping peptides recognized by the sera of macaques M308, M316, and M490 and M607 within the V1 region of SHIV_89.6P. (I): Sera (1:100 dilution) of the immunized macaques M218, M308, M316, and M490 before and after challenge exposure (weeks 10 and 19 and 2, 5 respectively) and the two control macaques. L915 and M320 were reacted with overlapping peptides 16–28 for the SF162 V1 region.

Figure 2. Immune responses in the vaccinated/challenged macaques

(A) ELISPOTs were performed using HIV_Ba-L, HIV_SF162, and HIV_89.6 Tev peptide pools for the stimulation of macaque blood. ELISPOT responses were also obtained using Concanavalin A, a stimulus to control for cell viability (data not shown). Antibody titers to Tev (B), Tat(C), Rev(D), and the pooled HIV_Ba-L, SHIV_SF162P3, and SHIV_89.6P Envelope proteins (E). The arrows refer to DNA immunizations (weeks 0, 4, 8), protein boosts (weeks 12, 18), and challenge exposure (week 22); titers to the purified Tev, Tat, Rev, and pooled HIV_Ba-L, HIV_SF162P3, and HIV_89.6P Envelope proteins. The weeks 0, 2, 6, and 10 refer to the weeks post challenge (p.c.). Data from control macaques are not shown. (F) Overlapping peptides of the entire HIV_89.6 Tev protein whose sequence is depicted in Fig. 1B were used with the sera (1:100 dilution) of the macaques after immunization (weeks 10, 19) and challenge exposure (weeks 2, 5 p.c.) in some of the vaccinated and control macaques. The area included in the dotted line area defines the V1 peptides (16 to 23), proceeded by the peptides derived from the first exon of Tat and followed by the peptides derived from the second exon of Rev. (G) Sera (1:100 dilution) of all immunized macaques after challenge exposure (weeks 2, 5 p.c.) and the two control macaques L915 and M320 were reacted with overlapping peptides 16–28 for the 89.6 V1. (H) Amino acid sequences of the 21-24 overlapping peptides recognized by the sera of macaques M308, M316, and M490 and M607 within the V1 region of SHIV_89.6P. (I): Sera (1:100 dilution) of the immunized macaques M218, M308, M316, and M490 before and after challenge exposure (weeks 10 and 19 and 2, 5 respectively) and the two control macaques. L915 and M320 were reacted with overlapping peptides 16–28 for the SF162 V1 region.

Figure 3. Viral variants within the V1 region following challenge exposure to SHIV_89.6P

Putative amino acid sequence of the SHIV_89.6P V1 region obtained by DNA sequencing of plasma viral RNA following RT/PCR. The putative amino acid sequences of V1 in the vaccinated (A) and control (B) macaques were aligned with the V1 region of the SHIV_89.6 used in the Tev vaccine. The number of viral clones sequenced is depicted on the left of each figure.

Figure 4. In vitro replication and sensitivity to ADCVI of SHIV_89.6 mutant viruses

The supernatant of 293T-cells transfected with the HIV_89.6WT, HIV_89.6L, and HIV_89.6LN mutants was normalized for p27Gag content and applied to TZM cells, CEMx174 cells, or primary human and rhesus PBMCs. Virus production was measured as activation of the HIV-! LTR Luciferase reporter gene in the TZM cells (A) or as p27Gag production in the supernatant fluid within the first 4 days of infection of the CEMx174 T-cells (B) or in human PBMCs up to day six post-infection (C).

Figure 5. Sensitivity to ADCVI of SHIV _89.6 viral variants

SHIV_89.6P-infected human CD4⁺ lymphocyte target cells were incubated with human PBMC effector cells (E:T = 10:1) and with plasma at the dilutions indicated. Eight days later, supernatant fluid p27Gag was determined by ELISA, and virus inhibition was determined as described in the Methods. Data are representative of several experiments, each performed in duplicate. Antibody-dependent cell-mediated virus inhibition (ADCVI) activity in plasma (1:100 dilution) of the animals was performed at first using the SHIV_89.6P before immunization week -2 (data not shown), or at time of challenge (A), or thereafter at week 4 (B) and 6 (C). ADCVI titers to the SHIV_89.6 viral variants a week 5 (D) and 22 (E) post challenge.

Figure 6. Neutralizing antibody titers and passive transfer of purified Ig to a naïve macaque

Neutralization antibody titers were measured as the highest serum/plasma dilution that induced a 50% reduction in luciferase reporter gene expression after a single round of infection in TZM-bl cells as described [38;39] using HIV _NL-ADArs(A) SF_162.LS, (B) or SHIV_89.6P. (C) The data presented were obtained with plasma collected at week 4 and 6 post challenge. (D) Titers of ADCVI in macaque M316 at 24 weeks post exposure to SHIV_89.6P. (E) Virus plasma levels in macaques challenged with SHIV_89.6P following treatment with purified Ig from macaques M316 (macaque P155) or normal human Ig (macaque P156).