Systemic immunization with an ALVAC-HIV-1/protein boost vaccine strategy protects rhesus macaques from CD4+ T-cell loss and reduces both systemic and mucosal simian-human immunodeficiency virus SHIVKU2 RNA levels

Abstract

Transmission of human immunodeficiency virus type 1 (HIV-1) occurs primarily via the mucosal route, suggesting that HIV-1 vaccines may need to elicit mucosal immune responses. Here, we investigated the immunogenicity and relative efficacy of systemic immunization with two human ALVAC-HIV-1 recombinant vaccines expressing Gag, Pol, and gp120 (vCP250) or Gag, Pol, and gp160 (vCP1420) in a prime-boost protocol with their homologous vaccine native Env proteins. The relative efficacy was measured against a high-dose mucosal exposure to the pathogenic neutralization-resistant variant SHIV(KU2) (simian-human immunodeficiency virus). Systemic immunization with both vaccine regimens decreased viral load levels not only in blood but unexpectedly also in mucosal sites and protected macaques from peripheral CD4+ T-cell loss. This protective effect was stronger when the gp120 antigen was included in the vaccine. Inclusion of recombinant Tat protein in the boosting phase along with the Env protein did not contribute further to the preservation of CD4+ T cells. Thus, systemic immunization with ALVAC-HIV-1 vaccine candidates elicits anti-HIV-1 immune responses able to contain virus replication also at mucosal sites in macaques.

FIG.1.

(a) Expression of HIV-1_IIIB Env proteins in chicken embryo fibroblasts infected with vCP 1420 and vCP250 ALVAC-HIV-1 vaccines. Chicken embryo fibroblasts were infected with vCP1420 (lanes 1 to 3), vCP250 (lanes 4 to 6), and mock virus (lanes 7 to 9) and labeled with [³⁵S]methionine overnight. Cells (lanes 3, 6, and 9) and supernatants (lanes 1, 2, 4, 5, 7, and 8) were lysed and immunoprecipitated with normal human serum (lanes 2, 5, and 8) or serum from an HIV-1-infected individual (lanes 1, 3, 4, 6, 7, and 9) and analyzed by SDS-PAGE, as described in Materials and Methods. (b) PAGE profiles of Env and Tat proteins. Proteins were heated at 95°C for 5 min with SDS sample buffer under reducing conditions, electrophoresed on 10 to 20% gradient gel, and stained with Coomassie blue. Lane 1, gp140; lane 2, gp120; lane 3, Tat; lane 4, molecular weight marker. (c) Schematic representation of the study design. The asterisks designate the time of Tat administration in half of the animals in groups A, B, and C.

FIG.1.

(a) Expression of HIV-1_IIIB Env proteins in chicken embryo fibroblasts infected with vCP 1420 and vCP250 ALVAC-HIV-1 vaccines. Chicken embryo fibroblasts were infected with vCP1420 (lanes 1 to 3), vCP250 (lanes 4 to 6), and mock virus (lanes 7 to 9) and labeled with [³⁵S]methionine overnight. Cells (lanes 3, 6, and 9) and supernatants (lanes 1, 2, 4, 5, 7, and 8) were lysed and immunoprecipitated with normal human serum (lanes 2, 5, and 8) or serum from an HIV-1-infected individual (lanes 1, 3, 4, 6, 7, and 9) and analyzed by SDS-PAGE, as described in Materials and Methods. (b) PAGE profiles of Env and Tat proteins. Proteins were heated at 95°C for 5 min with SDS sample buffer under reducing conditions, electrophoresed on 10 to 20% gradient gel, and stained with Coomassie blue. Lane 1, gp140; lane 2, gp120; lane 3, Tat; lane 4, molecular weight marker. (c) Schematic representation of the study design. The asterisks designate the time of Tat administration in half of the animals in groups A, B, and C.

FIG. 2.

Humoral immune response in vCP 250 and vCP1420 ALVAC-immunized macaques. (a) Antibody titers to HIV-1_IIIB gp140 protein were measured by ELISA in group A, group B, and group C animals on designated weeks after immunization on weeks 26, 52, 78, 225, and 233. (b) Anti-Tat antibody titers in the serum were measured on weeks 223, 227, and 234 after Tat protein boost in ALVAC-gp120 and ALVAC-gp160 animals. The minimum dilution of serum tested in the assay was 1:25. Values are expressed as mean antibody titers ± the standard errors from six animals from each group. (c) Neutralization of SHIV_HXB2 was measured in MT2 cells in the presence of sera from groups A, B, and C macaques collected on weeks 28, 54, 80, and 234. The lowest dilution of serum tested in the assay was 1:10. The results are expressed as dilution of serum inhibiting 50% of infection in MT2 cells and are expressed as mean titers ± the standard errors from six animals of each group.

FIG. 2.

Humoral immune response in vCP 250 and vCP1420 ALVAC-immunized macaques. (a) Antibody titers to HIV-1_IIIB gp140 protein were measured by ELISA in group A, group B, and group C animals on designated weeks after immunization on weeks 26, 52, 78, 225, and 233. (b) Anti-Tat antibody titers in the serum were measured on weeks 223, 227, and 234 after Tat protein boost in ALVAC-gp120 and ALVAC-gp160 animals. The minimum dilution of serum tested in the assay was 1:25. Values are expressed as mean antibody titers ± the standard errors from six animals from each group. (c) Neutralization of SHIV_HXB2 was measured in MT2 cells in the presence of sera from groups A, B, and C macaques collected on weeks 28, 54, 80, and 234. The lowest dilution of serum tested in the assay was 1:10. The results are expressed as dilution of serum inhibiting 50% of infection in MT2 cells and are expressed as mean titers ± the standard errors from six animals of each group.

FIG. 3.

Plasma viral RNA load in ALVAC-immunized macaques challenged with SHIV_KU2 by the rectal route. Plasma virus load was compared between naive group E and SIV-gag-pol group D (a), control (groups D and E) and vCP250-immunized/gp120-boosted group A animals (b), control (groups D and E) and vCP1420-immunized/gp140-boosted groups B and C animals (c), and control (groups D and E) and vCP250-immunized/gp120-boosted group A animals plus vCP1420-immunized/gp140-boosted groups B and C animals (d). Values are expressed as log-transformed mean RNA loads ± the standard errors from animals from each group.

FIG. 4.

CD4⁺ T-cell counts in ALVAC-immunized and control macaques after challenge exposure to SHIV_KU2. Mean CD4⁺ T-cell counts were compared between control groups (groups D and E) and vCP250 group A (a) and vCP1420 groups B and C (b), respectively. Values on day 0 represent average CD4⁺ T-cell counts from two time points measured before challenge.

FIG. 5.

RNA virus levels in the tissues of macaques in groups A (vCP250), B and C (vCP 1420), and E (naive) at euthanasia. SIV RNA virus levels in tissues were compared between naive animals, vCP250 group A, vCP1420 group B, vCP1420 group C, and vCP172 group D separately.

FIG. 6.

SHIV_HXB2 neutralizing antibody titers in the sera of infected macaques. Neutralization of SHIV_HXB2 was measured in MT2 cells in the presence of sera from groups A, B, C, D, and naive macaques collected on 2, 4, and 8 weeks after rectal inoculation with SHIV_KU2. The lowest dilution of serum tested was 1:10. The results are expressed as dilutions of serum inhibiting 50% of infection in MT2 cells and are expressed as mean titers ± the standard errors from animals of each group.