Immunogenicity of a polyvalent HIV-1 candidate vaccine based on fourteen wild type gp120 proteins in golden hamsters

Total RNA was purified from syncytium and non-syncytium inducing co-cultures of 14 HIV-1 patients (Table 1). Amplification products corresponding to the full length of gp120 (1.6 kb) containing constant and hypervariable regions were generated by RT-PCR. The genes were completely sequenced and identified as subtype B. The V3 amino acid sequence of the amplified gp120s was compared with V3 subtypes B, C and A/E, point mutations as well as insertion and deletion mutations were detected (Fig 1). In addition, the phylogenetic relations between the 14 different gp120 sequences and HIV subtypes B, C and A/E were revealed and genetic diversity between clones was identified (Fig 2). Due to the limited number of nucleotides in the V3 region, the algorithm is applied to a longitudinal data set of the C2-V5 regions of HIV-1 gp120s using Neighbor Tree-Maker in the FASTA format. By this approach, epidemiological linkage was established between the HIV-1 gp120 clones and HIV-1 subtypes B, C and A/E. Most of the HIV-1 gp120 sequences were clustered from the HIV-1 subtype B isolate MN. This isolate was epidemiologically linked to the subtype A/E isolate 93TH975. The results revealed no particular homology between gp120 clones and HIV-1 subtype C isolate 96ZM651. Each gp120 variant was cloned into the pEF6-myc-His vector for the expression and purification of recombinant gp120 proteins in vitro. Stable CHO cell lines transfected with the gp120 constructs selected for using blasticidine resistance and recombinant gp120 proteins were purified by metal affinity chromatography. The expression of all recombinant HIV-1 gp120 proteins has been determined by western blotting (Fig 3).

Four groups of hamsters were immunized with different vaccine formulations as indicated in Table 2. In the first group (A), hamsters were immunized with MPL adjuvant alone. In the second group (B), hamsters were immunized with 100 μg of the recombinant gp120 protein purified from patient number 001 plus adjuvant. Hamsters in the third group (C) were immunized with a mixture of proteins purified from patients #001 to #004 (25 μg of each recombinant gp120 protein) and adjuvant. The polyvalent group (D) immunized with a cocktail of all 14 proteins (~7 μg of each recombinant gp120 protein). Hamsters were immunized subcutaneously (S.C.) on weeks 0, 4, and 8. Animals were rested for 8 weeks and then boosted at week 16 with the same combination of gp120 proteins. Antibody titers were determined two weeks after each immunization.

In order to measure antibody titers against various HIV-1 gp120 proteins, plasma from immunized hamsters was collected two weeks after each immunization and analyzed by ELISA. The plates were coated with HIV-1 gp120 subtypes B (MN and SF162), C (96ZM651), and A/E (93TH975) overnight at 4°C followed by incubation with serially diluted plasma and developed as described in the Materials and Method section. The first two immunizations elicited a low level of antibody response to gp120 proteins (data not shown). However, after the third immunization, antibodies were detectable in all groups immunized with gp120. As expected the control group did not produce an antibody response. The highest level of antibody titer was observed in hamsters that were immunized with 14 recombinant gp120 proteins. In contrast, the control group (adjuvant alone) did not develop detectable levels of specific antibody responses. Eight weeks after the third immunization, animals were vaccinated again with respective recombinant gp120 proteins. Results similar to the third immunization were observed against gp120 subtypes between the groups. The polyvalent vaccine group induced higher levels of anti-gp120 titers to MN and SF162 (subtypes B) than the subtypes C and A/E (Fig 4). These results demonstrate that the polyvalent vaccine induces a higher antibody response to subtype B than the single gp120 candidate vaccine.

Two weeks after the fourth immunization, sera were collected and Nabs to HIV-1 strains were tested. Unlike many research studies, to detect the real breadth and strength of candidate vaccines, we used different HIV-1 strains, to assay for Nabs, from those, which were used in immunization. Since some background was observed in the pre-bleed samples, the Nab responses were compared with SVA-MLV (murine retrovirus Env-pseudovirus) as a background control for non-specific activity. The group of hamsters that received all 14 gp120 proteins showed higher Nabs titres to the HIV-1 MN strain than the single gp120 (p < 0.05), however, similar Nabs titres were observed in the group immunized with a combination of four gp120 proteins (Fig 5). Sera from immunized hamsters were further tested against other primary HIV-1 isolates but none of the sera were able to show significant neutralization.

The proliferation of CD4+T cells plays an important role in both humoral and cellular immune responses by expansion of antigen-stimulated B and CD8+T cells, respectively. A high proliferative immune response was observed to the gp120 protein isolated from patient #001 in all groups with the exception of the control group (data not shown). However, to assess and measure the strength of the candidate vaccines, splenocytes from immunized hamsters were further stimulated with HIV-1 gp120 strains and subtypes that were not included in the vaccine components. Splenocytes were cultured in the presence of HIV-1 gp120 proteins. Two days after stimulation, [³H] thymidine was added and sixteen hours later, cells were harvested and thymidine incorporation was measured. The groups B and C showed a relatively similar proliferative immune response. Interestingly, the group of hamsters that was immunized with all 14 gp120 proteins showed the highest lymphocyte proliferative immune response (Fig 6) to subtypes B (MN and SF162), C (96ZM651) and A/E (93TH975), supporting the cross-subtype activity of Th immune responses.

Viruses were isolated from HIV-infected individuals during their visits to the HIV clinic in the Ottawa Hospital. Verbal consent was obtained from HIV patients for participation in the study. Briefly, isolation of virus was performed by placing 2–4 × 10⁶ peripheral blood mononuclear cells (PBMCs) in Iscove's Modified Dulbecco's Medium (IMDM: Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (FCS: Life Technologies, Grand Island, NY), 2 Mm Glutamine (Sigma) and 50 μg/ml gentamicine (Sigma) with an equal number of phytohemagglutinin (PHA: Sigma) stimulated donor cells. The cells were incubated at 37°C containing 5% CO₂ and 20 IU/ml Interleukin-2 (IL-2: R&D systems, Minneapolis, MN) was added to increase the growth of these cells. Co-cultures were maintained for up to 4 weeks with periodic media changes. 2 × 10⁶ freshly stimulated donor cells were added every 10 days to sustain HIV replication. Viruses were measured in the supernatant by HIV p24 antigen kit (DuPont, Wilmington, DE).

Total RNA was purified using RNeasy extraction kit (Qiagen, Mississauga, ON) from syncytium and non-syncytium inducing HIV-1 subtype B co-cultures. The encoding gp120 sequences (1.6 kb) were amplified by RT-PCR. The amplicons were purified using the QIAquick gel extraction kit (Qiagen, Mississauga, ON) and cloned into the PCR 2.1 TOPO-TA vector (Invitrogen, Burlington, ON). After plasmid digestion, the 1.6 kb band corresponding to the gp120 genes were sub-cloned into the pEF6-myc-His vectors (Invitrogen) which is designed for the over-production of recombinant proteins in mammalian cell lines. In this system, recombinant proteins may be purified by affinity chromatography using the c-myc epitope or poly histidine (6 × His) metal-binding tags. The expression constructs were confirmed and characterized by restriction enzymes and nucleotide sequence analysis.

All fourteen HIV-1 gp120-V3 regions were aligned using "ClustalW" which is a multiple sequence alignment program for DNA or proteins [39, 40]. At the end, the sequences were also manually adjusted. The phylogenetic tree was constructed by the Neighbor Tree-Maker in HIV sequence database. The program converts sequences alignment to the FASTA format.

CHO cells were grown at 37°C, 5% CO₂ in IMDM (Sigma) supplemented with 10% FCS (Life Technologies), 100 U/ml penicillin and 100 μg/ml gentamicin (Sigma).

CHO cells were transfected with pEF6-myc-His vectors expressing HIV-1 gp120 structural genes. Stable cell lines were established by using blasticidine-supplemented medium. Cells were harvested, sonicated and lysed in lysis buffer (25 mM Tris base, 2.5 mM Mercaptoethanol, 1% Triton-X100 and a cocktail of protease inhibitors). Recombinant viral proteins were purified with the TALON metal resin kit (Clontech, Palo Alto, CA) as per manufacturer's instructions. The recombinant proteins were confirmed by western blotting and Immunofluorecence antibody staining as previously described [41‐43].

Six-week old golden hamsters were purchased from Jackson Laboratory (Bar Harbor, ME). Hamsters were maintained in accordance with laboratory standard procedures and were housed in Animal Care in the Faculty of Medicine at University of Ottawa. A total of 100 μg recombinant protein in 0.2 mg of Monophosphoryl lipid A (MPL: Corixa, Hamilton, MT) was injected per animal. Groups of four hamsters were immunized four times subcutaneously (S.C.) in three different sites with one month interval between each immunization. Fourteen days after the last boost, the hamsters were sacrificed and their spleens and blood were collected for further testing or long-term storage in cryopreservation medium.

96-well ELISA plates were coated overnight at 4°C with gp120 proteins clade B (MN, SF162), C (96ZM651) and A/E (93TH975) obtained from the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases (NIAID). The plates were washed with PBS containing 0.05% Tween 20 and then blocked for 1 h with 1% BSA in PBS. Following a second wash with PBS/0.05% Tween 20, the plates were incubated for 2 h at 37°C with serially diluted sera. The plates were washed and incubated for 2 h with peroxidase-conjugated affinity-purified rabbit anti-hamster secondary antibody (Bio-Rad). After washing, color was developed with O-phenylendiamine dihydrochloride (OPD: Sigma, St. Louise, MO). The color reaction was stopped with 1 N HCl and absorbance was read at 490 nm with an ELISA plate reader (Bio-Rad).

Neutralization was measured as a function of reduction in luciferase reporter gene expression after a single round of infection in TZM-bl cells. TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program, as contributed by John Kappes and Xiaoyun Wu. Briefly, 200 TCID50 of virus was incubated with serial 3-fold dilutions of serum sample in triplicate in a total volume of 150 μl for 1 hr at 37°C in 96-well flat-bottom culture plates. Freshly trypsinized TZM-bl cells (10,000 cells in 100 μl of growth medium containing 75 μg/ml DEAE dextran) were added to each well. Indinavir was added at a final concentration of 1 μM to prevent virus replicaton in the case of HIV-1 MN. One set of control wells received cells + virus (virus control) and another set received cells only (background control). After a 48 hour incubation, 100 μl of cells was transferred to 96-well black solid plates (Corning Costar, Rochester, NY) for measurement of luminescence using Bright Glo substrate solution as described by the supplier (Promega, Madison WI). An assay stock of HIV-1 MN was prepared in H9 cells. Assays stocks of Env-pseudotyped viruses were prepared by tranfection in 293T cells. All virus stocks were made cell-free by 0.45-micron filtration and were stored at -70°C until use.

The assay was performed by [³H] thymidine incorporation as previously described [44]. Briefly, splenocytes from immunized hamsters were resuspended at 2 × 10⁶ cells/ml in RPMI 1640 containing 10% FCS, 50 μM β-mercaptoethanol and 100 U/ml penicillin/streptomycin. A 100 μl aliquot containing 2 × 10⁵ cells was added to each well of a 96 well plate. The gp120 protein subtypes B, C and A/E (100 μl at 20 μg/ml) were added to each well in triplicate. As a positive control, cells were also stimulated with phorbol 12-myristate 13-acetate and ionomycin (PMA/ION: Sigma). After 48 h of culture, 1 μCi [³H] thymidine (Amersham, Arlington Heights, IL) was added to each well. Following 16 h of incubation, cells were harvested onto glass fibre filtermats and thymidine incorporation was measured with a Microbeta beta counter (Wallac, Turku, Finland).