The ability of an oligomeric human immunodeficiency virus type 1 (HIV-1) envelope antigen to elicit neutralizing antibodies against primary HIV-1 isolates is improved following partial deletion of the second hypervariable region

Abstract

Partial deletion of the second hypervariable region from the envelope of the primary-like SF162 virus increases the exposure of certain neutralization epitopes and renders the virus, SF162DeltaV2, highly susceptible to neutralization by clade B and non-clade B human immunodeficiency virus (HIV-positive) sera (L. Stamatatos and C. Cheng-Mayer, J. Virol. 78:7840-7845, 1998). This observation led us to propose that the modified, SF162DeltaV2-derived envelope may elicit higher titers of cross-reactive neutralizing antibodies than the unmodified SF162-derived envelope. To test this hypothesis, we immunized rabbits and rhesus macaques with the gp140 form of these two envelopes. In rabbits, both immunogens elicited similar titers of binding antibodies but the modified immunogen was more effective in eliciting neutralizing antibodies, not only against the SF162DeltaV2 and SF162 viruses but also against several heterologous primary HIV type 1 (HIV-1) isolates. In rhesus macaques both immunogens elicited potent binding antibodies, but again the modified immunogen was more effective in eliciting the generation of neutralizing antibodies against the SF162DeltaV2 and SF162 viruses. Antibodies capable of neutralizing several, but not all, heterologous primary HIV-1 isolates tested were elicited only in macaques immunized with the modified immunogen. The efficiency of neutralization of these heterologous isolates was lower than that recorded against the SF162 isolate. Our results strongly suggest that although soluble oligomeric envelope subunit vaccines may elicit neutralizing antibody responses against heterologous primary HIV-1 isolates, these responses will not be broad and potent unless specific modifications are introduced to increase the exposure of conserved neutralization epitopes.

FIG. 1

Development of antibodies in rabbits. Six animals (A1 to A6) were immunized with DNA expressing the unmodified SF162 gp140 immunogen, and six (A7 to A12) were immunized with DNA expressing the modified ΔV2 gp140 immunogen. Titers were determined 2 weeks following each immunization, as described in Materials and Methods, against the oligomeric SF162 and ΔV2 gp140 proteins. Dashed lines indicate times of immunizations.

FIG. 2

Results of neutralization experiments using rabbit sera collected following the third and fifth immunizations against the SF162ΔV2 (A) and SF162 (B) viruses. Data are representative of at least three independent experiments. Symbols indicate the mean percent neutralization and standard deviation from triplicate wells. Dashed lines indicate 50, 70, and 90% inhibition of infection. Asterisks (controls) represent neutralization curves obtained with sera collected from animals that were immunized with the DNA vector alone and are indicative of nonspecific neutralization.

FIG. 2

Results of neutralization experiments using rabbit sera collected following the third and fifth immunizations against the SF162ΔV2 (A) and SF162 (B) viruses. Data are representative of at least three independent experiments. Symbols indicate the mean percent neutralization and standard deviation from triplicate wells. Dashed lines indicate 50, 70, and 90% inhibition of infection. Asterisks (controls) represent neutralization curves obtained with sera collected from animals that were immunized with the DNA vector alone and are indicative of nonspecific neutralization.

FIG. 3

Generation of antibodies in rhesus macaques. The generation of antienvelope antibodies in two animals (J408 and H445) immunized with the modified ΔV2gp140 immunogen and two animals (P655 and N472) immunized with the unmodified SF162gp140 immunogen, as well as control animals (M844 and H473) immunized with the DNA vector alone, were determined by ELISA using the corresponding protein as described in Materials and Methods. Dashed lines indicate times of immunizations. DNA, animals received three monthly immunizations with DNA vectors expressing the gp140 form of each immunogen. Control animals received the DNA vector alone. DNA plus Protein, animals received a fourth DNA immunization and at the same time were immunized with the corresponding CHO cell-produced oligomeric gp140 proteins, mixed in the adjuvant MF-59C. Control animals received adjuvant alone.

FIG. 4

Neutralizing activities of rhesus macaque sera. Neutralization activities against the SF162 and SF162ΔV2 viruses of sera collected from animals immunized with the modified ΔV2 gp140 (A) and the unmodified (B) SF162 gp140 immunogens were determined as described in Materials and Methods. Dashed lines indicate 50, 70, and 90% inhibition of infection. Results are representative of three to five independent experiments. Data indicate the mean and standard deviation from triplicate wells. Pre-bleeds, sera collected before vaccination; 2nd DNA and 3rd DNA, sera collected 1 month following the second and the third, respectively, DNA administrations; 2 and 4 weeks post 1st boost, sera collected 2 and 4 weeks, respectively, following the DNA-plus-protein booster immunization.

FIG. 4

Neutralizing activities of rhesus macaque sera. Neutralization activities against the SF162 and SF162ΔV2 viruses of sera collected from animals immunized with the modified ΔV2 gp140 (A) and the unmodified (B) SF162 gp140 immunogens were determined as described in Materials and Methods. Dashed lines indicate 50, 70, and 90% inhibition of infection. Results are representative of three to five independent experiments. Data indicate the mean and standard deviation from triplicate wells. Pre-bleeds, sera collected before vaccination; 2nd DNA and 3rd DNA, sera collected 1 month following the second and the third, respectively, DNA administrations; 2 and 4 weeks post 1st boost, sera collected 2 and 4 weeks, respectively, following the DNA-plus-protein booster immunization.

FIG. 5

Neutralization of heterologous clade B primary HIV-1 isolates by macaque sera. Neutralization activities of sera collected 2 and 4 weeks following the DNA-plus-protein booster immunization against isolates heterologous to the vaccine primary HIV-1 isolates were determined as described in Materials and Methods. Dashed lines indicate 50, 70, and 90% inhibition of infection. The values represent specific neutralization, which is defined as the difference between the percent virus neutralization recorded with sera collected following vaccination and that recorded with sera collected prior to the initiation of vaccination. Data points indicate the mean percent specific neutralization from two independent experiments.

FIG. 6

Generation of binding and neutralizing antibodies following the second booster immunization with the modified ΔV2 gp140 protein. (A) The generation of antienvelope antibodies in two rhesus macaques (J408 and H445) vaccinated with the modified ΔV2 gp140 immunogen was determined by ELISA as described in Materials and Methods. Dashed lines indicate times of immunizations. DNA, animals received three monthly immunizations with DNA vectors expressing the gp140 form of this immunogen; DNA plus Protein, animals received a fourth DNA immunization and purified oligomeric ΔV2 gp140 protein; Protein, the animals were immunized with the purified oligomeric ΔV2 gp140 protein alone. (B) Neutralization activities against the SF162ΔV2 and SF162 isolates of sera following the second boost were compared to those of sera collected following the first boost (see also Fig. 4). Nonspecific neutralization recorded with preimmunization sera (Pre-bleeds) is also shown.

FIG. 7

Presence of anti-V3 loop antibodies in sera collected from macaques immunized with the modified ΔV2 gp140 immunogen. The development of anti-V3 loop antibodies was determined by ELISA using the V3 loop peptide derived from the SF162/SF162ΔV2 envelope. (A) We first examined whether the captured V3 loop peptide interacts with specific anti-V3 loop MAbs recognizing linear (447D) and conformational (391-95D) V3 loop epitopes. (B) We next determined the titer of anti-V3 loop antibodies present in sera collected 2 and 4 weeks following the 1^st and 2^nd boosts from the two vaccinated animals. As a comparison, we also include the titers of total antienvelope antibodies present in the same sera.