Development of a single-shot subunit vaccine for HIV-1. 5. programmable in vivo autoboost and long lasting neutralizing response.

The subunit vaccine for HIV-1, recombinant glycoprotein 120 (rgp120), was used as a model antigen to evaluate the potential for a pulsatile single immunization vaccine formulation consisting of poly(lactic-co-glycolic) acid (PLGA) microspheres. We designed rgp120 PLGA microsphere formulations that provide a pulse of rgp120 at 1 to 6 months (depending on the polymer) after administration, mimicking another immunization. In these studies, the in vitro pulse of rgp120 correlated well with the observed in vivo autoboost as measured by an increase in anti-gp120 antibodies in guinea pigs. The immune response to the rgp120 PLGA microsphere formulations was increased by adding the soluble form of the saponin-derived adjuvant, QS-21. The use of small microspheres, however, did not increase the humoral response to rgp120. A single immunization with rgp120 PLGA microspheres resuspended in soluble rgp120 and QS-21 elicited neutralizing antibody titers that were comparable to titers obtained from two immunizations of rgp120 and QS-21 at the same total dose. Administration of rgp120 PLGA microspheres in baboons resulted in high, long-lasting neutralizing antibody titers that were greater than repeated immunizations with soluble rgp120 and QS-21. These studies also indicated that a continuous release of QS-21 at the injection site may provide a greater immune response than a bolus injection. Overall, this work demonstrated that PLGA microsphere formulations may be designed to provide in vivo pulses of an antigen eliminating the need for repeated immunizations.

Genetic and immunologic characterization of viruses infecting MN-rgp120-vaccinated volunteers.

Proviral sequences were determined and immunologic characterization was carried out for envelope glycoproteins from 7 vaccinees who became infected with human immunodeficiency virus type 1 (HIV-1), through high-risk behavior, while participating in clinical trials of MN-rgp120, a candidate HIV-1 vaccine. All 7 infections resulted from subtype B viruses; however, only 3 of the viruses possessed the MN serotype-defining V3 domain sequence, IGPGRAF, prevalent in 60%-70% of US infections. Six of the 7 viruses differed from MN-rgp120 at a neutralizing epitope in the C4 domain, and all 7 differed from MN-rgp120 at a neutralizing epitope in the V2 domain. Recombinant gp120 was prepared from each breakthrough specimen and tested for binding to a panel of neutralizing monoclonal antibodies. The results suggest that 6 of 7 breakthrough infections may be related to incomplete immunization or to infection with viruses that differed from the vaccine immunogen at important virus-neutralizing epitopes.

Protection of MN-rgp120-immunized chimpanzees from heterologous infection with a primary isolate of human immunodeficiency virus type 1.

Three chimpanzees immunized with recombinant gp120 from human immunodeficiency virus type 1 (HIV-1) strain MN and 1 control animal were challenged intravenously with a primary isolate of HIV-1SF2. Viral infection was detected in the control animal by viral culture, polymerase chain reaction, and multiple serologic assays beginning 2 weeks after infection. Markers of HIV-1 infection were not detected in any of the gp120-vaccinated animals during 12 months of follow-up. Antisera from the gp120-immunized chimpanzees were unable to neutralize the challenge virus cultured in peripheral blood mononuclear cells (PBMC). These studies demonstrate that immunization with recombinant gp120 derived from a T cell-adapted isolate prevented infection by a heterologous primary isolate of HIV-1. The results suggest that in vitro virus neutralization assays utilizing primary isolates cultured in PBMC may be imperfect indicators of protection in vivo.

Effect of adjuvants on immunogenicity of MN recombinant glycoprotein 120 in guinea pigs.

The immunogenicity of recombinant gp120 from the MN strain of HIV-1, a candidate HIV-1 vaccine, was evaluated in guinea pigs using adjuvant formulations with different physical and chemical properties. The adjuvants tested included Freund's adjuvant (FA), alum, and the novel adjuvant QS-21. These studies demonstrated that QS-21 provides a number of advantages compared to the two other adjuvants tested. QS-21 formulations accelerated the production of antibodies to MN rgp120 and elicited complete seroconversion after a single immunization. QS-21 shifted the antigen dose-response curve for antibody production by as much as three orders of magnitude, enabling a more economical use of antigen. Antibody titers to MN rgp120 and to the principal neutralizing determinant in the V3 domain were higher in animals receiving QS-21 formulations than in animals immunized with the other adjuvants, and correlated well with higher virus neutralization titers in an in vitro assay. These results support the testing of QS-21 in future clinical trials of candidate HIV-1 vaccines.

Comparison of the immune response to recombinant gp120 in humans and chimpanzees.

OBJECTIVE: To assess similarities and differences in antibody responses to recombinant (r) HIV-1IIIB gp120 in chimpanzees, previously protected from HIV-1 infection, and human volunteers immunized in connection with a Phase I clinical trial. METHODS: Frozen sera from humans immunized with rgp120 from HIV-1IIIB and chimpanzees immunized with the same antigen or recombinant soluble gp160 were compared in a variety of serologic assays. RESULTS: The magnitude of the antibody response to gp120 was similar in both species; however, the half-life of the antibody response to rgp120 was approximately 4.5 times longer in humans (9 weeks) than in chimpanzees (2 weeks). Antibodies to gp120 in both species were broadly cross-reactive with gp120 from diverse isolates of HIV-1 and were effective in blocking the binding of gp120 to CD4. Antibody binding to native gp120 was greater than to denatured gp120 in both species. Antibody responses to the principal neutralizing determinant (V3 domain) and virus neutralization titers were approximately 10-fold lower in humans than chimpanzees. The relative avidity of antibody binding to gp120 was higher in the sera from the immunized chimpanzees than in the immunized humans. CONCLUSIONS: While the antibody responses to rgp120 elicited in man and chimpanzees were in many ways similar, significant differences did occur. Predictions made on the basis of chimpanzee immunogenicity studies over-estimated the potency of the virus neutralizing titers and under-estimated the duration of the antibody response achieved in humans.

Immunogenicity and HIV-1 virus neutralization of MN recombinant glycoprotein 120/HIV-1 QS21 vaccine in baboons .

The effect of adjuvant and immunization schedule on the immunogenicity of HIV-1 envelope glycoprotein, MN rgp120, was optimized by using baboons. The novel adjuvant QS21 elicited earlier seroconversion than alum adjuvant, and the antibody titers to MN rgp120 for animals treated with QS21 were significantly greater than the titers obtained in animals treated with alum. The use of QS21 shifted the dose-response curve, resulting in less MN rgp120 required to achieve equivalent titers to those in the alum formulations. The in vitro virus neutralizing (VN) titers from animals treated with QS21 were 3- to 10-fold higher than with alum. The data presented herein point to the superiority of QS21 as adjuvant in primates for MN rgp120.

Development of a single-shot subunit vaccine for HIV-1.

The successful development of an AIDS vaccine will require formulations that not only invoke the desired immunological response, but also are stable and easy to administer. A single shot MN rgp120 vaccine formulation comprised of MN rgp120 encapsulated in poly (lactic-coglycolic) acid (PLGA) microspheres was developed to provide an in vivo autoboost of antigen. These formulations were designed to yield an in vivo autoboost at 1, 2, 3 or 4-6 months. In addition, PLGA microspheres containing the adjuvant, QS21, were also prepared to provide an in vivo autoboost concomitant with antigen. In guinea pigs, these formulations yielded higher anti-MN rgp120 and anti-V3 loop antibody titers than alum formulations that were administered at higher antigen doses. Different doses of encapsulated MN rgp120 provided a clear and well-defined dose response curve for both anti-MN rgp120 and anti-V3 loop antibody titers. When soluble QS21 was mixed with the encapsulated MN rgp120, the antibody titers were increased by a factor of 5 over the titers with encapsulated MN rgp120 alone. An additional fivefold increase in antibody titers was observed for guinea pigs immunized with encapsulated MN rgp120 and QS21 on the same microspheres. These results suggest that the adjuvant properties of QS21 can be increased by microencapsulation in PLGA. Furthermore, antibodies induced by these preparations neutralized the MN strain of HIV-1. The neutralization titers for sera from animals immunized with MN rgp120-PLGA and soluble QS21 were greater than the titers obtained from guinea pigs that were treated with MN rgp120 and soluble QS21 at the same dose. Overall, these studies validate the in vivo autoboost concept, reveal a method for improving the adjuvant properties of QS21, and indicate the potential of future single shot vaccine formulations.

Monoclonal antibodies to the extracellular domain of HIV-1IIIB gp160 that neutralize infectivity, block binding to CD4, and react with diverse isolates.

Ten monoclonal antibodies prepared against a soluble, recombinant form of gp160, derived from the IIIB isolate of HIV-1, were characterized. Four of the antibodies neutralized HIV-1IIIB infectivity in vitro, three blocked the binding of recombinant gp120 to CD4, three were reactive with gp41, and one preferentially reacted with an epitope on gp120 within the gp160 precursor. All three CD4 blocking antibodies bound to distinct epitopes, with one mapping to the C1 domain, one mapping to the C4 domain, and one reactive with a conformation-dependent, discontinuous epitope. Of these, the antibody reactive with the discontinuous epitope exhibited neutralizing activity against homologous and heterologous strains of HIV-1. The binding of these monoclonal antibodies to a panel of seven recombinant gp120s prepared from diverse isolates of HIV-1 was measured, and monoclonal antibodies with broad cross reactivity were identified. The epitopes recognized by 7 of the 10 monoclonal antibodies studied were localized by their reactivity with synthetic peptides and with fragments of gp120 expressed as fusion proteins in a lambda gt-11 gp160 epitope library.

Neutralization of multiple laboratory and clinical isolates of human immunodeficiency virus type 1 (HIV-1) by antisera raised against gp120 from the MN isolate of HIV-1.

Vaccines prepared from the envelope glycoprotein, gp120, of the common laboratory isolate of human immunodeficiency virus type 1 (HIV-1) (IIIB/LAV-1) elicit antibodies that neutralize the homologous virus but show little if any cross-neutralizing activity. This may be because the principal neutralizing determinant (PND) of gp120 is highly unusual in the IIIB/LAV-1 strain and is not representative of those found in the majority of field isolates. We have now examined the immunogenicity of recombinant gp120 prepared from the MN strain of HIV-1 (MN-rgp120), whose PND is thought to be representative of approximately 60% of the isolates in North America. Our results show that MN-rgp120 is a potent immunogen and elicits anti-gp120 titers comparable to those found in HIV-1-infected individuals. While both MN-rgp120 and IIIB-rgp120 induced antibodies able to block gp120 binding to CD4, strain-specific and type-common blocking antibodies were detected. Finally, antibodies to MN-rgp120 but not to IIIB-rgp120 were effective in neutralizing a broad range of laboratory and clinical isolates of HIV-1. These studies demonstrate that susceptibility or resistance to neutralization by antibodies to gp120 correlates with the PND sequence and suggest that the problem of antigenic variation may not be insurmountable in the development of an effective AIDS vaccine.