Induction of humoral immune responses following vaccination with envelope-containing, formaldehyde-treated, thermally inactivated human immunodeficiency virus type 1.

The lack of success of subunit human immunodeficiency virus type 1 (HIV-1) vaccines to date suggests that multiple components or a complex virion structure may be required. We previously demonstrated retention of the major conformational epitopes of HIV-1 envelope following thermal treatment of virions. Moreover, antibody binding to some of these epitopes was significantly enhanced following thermal treatment. These included the neutralizing epitopes identified by monoclonal antibodies 1b12, 2G12, and 17b, some of which have been postulated to be partially occluded or cryptic in native virions. Based upon this finding, we hypothesized that a killed HIV vaccine could be derived to elicit protective humoral immune responses. Shedding of HIV-1 envelope has been described for some strains of HIV-1 and has been cited as one of the major impediments to developing an inactivated HIV-1 vaccine. In the present study, we demonstrate that treatment of virions with low-dose formaldehyde prior to thermal inactivation retains the association of viral envelope with virions. Moreover, mice and nonhuman primates vaccinated with formaldehyde-treated, thermally inactivated virions produce antibodies capable of neutralizing heterologous strains of HIV in peripheral blood mononuclear cell-, MAGI cell-, and U87-based infectivity assays. These data indicate that it is possible to create an immunogen by using formaldehyde-treated, thermally inactivated HIV-1 virions to induce neutralizing antibodies. These findings have broad implications for vaccine development.

Anti-IgE efficacy in murine asthma models is dependent on the method of allergen sensitization.

BACKGROUND: Murine models used to delineate mechanisms and key mediators of asthma have yielded conflicting results and suggest that the dominant mechanism and mediators required for disease induction differ depending on the model and method of allergen sensitization used. OBJECTIVE: The goal of this study was to determine whether the mode of allergen sensitization influenced the role that IgE had in allergen-induced pulmonary eosinophilic inflammation. METHODS: Mice were exposed to dust mite extract in 2 models of allergic inflammation that differed in the method of sensitization. We compared sensitization by aerosol exposure with and without concomitant human respiratory syncytial virus infection with sensitization by means of systemic (intraperitoneal) exposure with adjuvant. After sensitization, animals were similarly challenged with aerosolized allergen. Animals were treated with anti-IgE mAb to deplete IgE and to determine its role in the induction of allergic inflammation and mucosa pathology in these models. RESULTS: Concomitant respiratory syncytial virus infection significantly enhanced allergen sensitization by aerosol exposure and exacerbated eosinophilic inflammation and airway mucosa pathology. Depletion of IgE in this model significantly reduced lung eosinophilic inflammation and airway mucosa pathology. However, in the model in which animals were sensitized by means of systemic allergen exposure with adjuvant, depletion of IgE had no ameliorative effect on lung inflammation or pathology. CONCLUSION: We demonstrated that the method of antigen sensitization can delineate the role of IgE in allergen-induced lung inflammation. In a murine model that more closely resembles ambient allergen exposure in human subjects, IgE had a critical role in the pathogenesis of allergic asthma and mucosa pathology. The results parallel the results reported with anti-IgE efficacy in allergic asthmatic human subjects.

Virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in HIV-infected patients with detectable viremia.

BACKGROUND: In many patients with human immunodeficiency virus (HIV) infection, therapy with potent antiretroviral drugs does not result in complete suppression of HIV replication. The effect of cessation of therapy in these patients is unknown. METHODS: Sixteen patients who had a plasma HIV RNA level of more than 2500 copies per milliliter during combination antiretroviral-drug therapy were randomly assigned, in a 2:1 ratio, to discontinue or continue therapy. Plasma HIV RNA levels, CD4 cell counts, and drug susceptibility were measured weekly. Viral replicative capacity was measured at base line and at week 12. RESULTS: Discontinuation of therapy for 12 weeks was associated with a median decrease in the CD4 cell count of 128 cells per cubic millimeter and an increase in the plasma HIV RNA level of 0.84 log copies per milliliter. Virus from all patients with detectable resistance at entry became susceptible to HIV-protease inhibitors within 16 weeks after the discontinuation of therapy. Drug susceptibility began to increase a median of six weeks after the discontinuation of therapy and was temporally associated with increases in plasma HIV RNA levels and decreases in CD4 cell counts. Viral replicative capacity, measured by means of a recombinant-virus assay, was low at entry into the study and increased after therapy was discontinued. Despite the loss of detectable resistance in plasma, resistant virus was cultured from peripheral-blood mononuclear cells in five of nine patients who could be evaluated. Plasma HIV RNA levels, CD4 cell counts, and drug susceptibility remained stable in the patients who continued therapy. CONCLUSIONS: Despite the presence of reduced drug susceptibility, antiretroviral-drug therapy can provide immunologic and virologic benefit. This benefit reflects continued antiviral-drug activity and the maintenance of a viral population with a reduced replicative capacity.

A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1.

Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.

Development of bivalent (B/E) vaccines able to neutralize CCR5-dependent viruses from the United States and Thailand.

Recombinant envelope glycoproteins prepared from a subtype B (MN) strain and a subtype E (CM244) strain of HIV-1 were combined to create a bivalent vaccine (B/E) effective against viruses circulating in the United States and Asia. Combining the two antigens resulted in formulations that increased the breadth and potency of the inter-subtype neutralizing response. Antibodies to the bivalent vaccine formulation neutralized viruses possessing diverse phenotypes, including syncytia-inducing and non-syncytia-inducing primary isolates, viruses using either the CCR5 or the CXCR4 chemokine receptors, and viruses differing in their sensitivity to soluble CD4. These studies demonstrate for the first time that the magnitude and quality of the immune response to HIV-1 can be improved by combining recombinant envelope glycoproteins from different genetic subtypes.

Characterization of V3 loop-Pseudomonas exotoxin chimeras. Candidate vaccines for human immunodeficiency virus-1.

To develop a candidate vaccine for human immunodeficiency virus, type 1 (HIV-1), chimeric proteins were constructed by inserting sequences derived from the V3 loop of gp120 into a nontoxic form of Pseudomonas exotoxin (PE). Inserts of 14 or 26 amino acids, constrained by a disulfide bond, were introduced between domains II and III of PE. V3 loop-toxin proteins expressed in Escherichia coli and corresponding to either MN (subtype B) or Thai (subtype E) strains, were recognized by strain-specific monoclonal anti-gp120 antibodies. When loop sequences were introduced into an enzymatically active form of the toxin, there was no loss of toxin-mediated cell killing, suggesting that these sequences were co-transported to the cytosol. Sera from rabbits injected with nontoxic PE-V3 loop chimeras were reactive for strain-specific gp120s in Western blots, immunocapture assays, enzyme-linked immunosorbent assays, and neutralized HIV-1 infectivity. Since toxin vectors were designed to receive oligonucleotide duplexes encoding any V3 loop sequence, this approach should allow for the production of V3 loop-toxin chimeras corresponding to multiple HIV isolates.

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.

Inhibition of HIV type 1 infectivity by constrained alpha-helical peptides: implications for the viral fusion mechanism.

Linear peptides derived from the membrane proximal region of the gp41 ectodomain are effective inhibitors of HIV type 1 (HIV-1)-mediated fusion events. These inhibitory peptides lack structure in solution, rendering mechanistic interpretation of their activity difficult. Using structurally constrained analogs of these molecules, we demonstrate that the peptides inhibit infectivity by adopting a helical conformation. Moreover, we show that a specific face of the helix must be exposed to block viral infectivity. Recent crystal structures show that the region of gp41 corresponding to the inhibitory peptides is helical and uses the analogous face to pack against a groove formed by an N-terminal coiled-coil trimer. Our results provide a direct link between the inhibition of HIV-1 infectivity by these peptides and the x-ray structures, and suggest that the conformation of gp41 observed by crystallography represents the fusogenic state. Other agents that block HIV-1 infectivity by binding to this groove may hold promise for the treatment of AIDS.

Development of a single-shot subunit vaccine for HIV-1. 2. Defining optimal autoboost characteristics to maximize the humoral immune response.

The design of a single-shot subunit vaccine for HIV-1 with polylactic-coglycolic acid (PLGA) sustained-release technology to effect an autoboost of antigen (MN gp120) at a given time after the primary immunization requires in-depth knowledge about the timing, the duration, and the need for coadjuvant in the autoboost. These questions cannot be answered unambiguously with PLGA microspheres, so we have conducted studies using Alzet minipumps to release antigen at prescribed times to mimic a PLGA autoboost. The results show that a discrete autoboost is preferred over continuous release of antigen, that the time profile of the autoboost (whether pulsatile or a 2-week continuous release) does not affect the booster immune response, and that only antigen is required in the booster immunization (a coadjuvant in the boost does not give higher titers).

Development of a single-shot subunit vaccine for HIV-1. 3. Effect of adjuvant and immunization schedule on the duration of the humoral immune response to recombinant MN gp120.

HIV-1 prophylaxis may require "sterilizing immunity" (i.e., the prevention of infection), and this is likely to demand a vaccine that gives high, long-lasting antibody titers. Although it is known that vaccine adjuvants and immunization schedule affect the magnitude of the immune response, there are few reports on antibody decay rates and persistence. Guinea pigs were immunized with recombinant gp120 using different adjuvants and immunization schedules, and the anti-gp120 and HIV-1 neutralization titers were determined over time following the last booster immunization. As observed previously in the literature, a longer time between boosting gave higher titers, with a slight increase in the decay half-life as the booster was spaced farther out from the primary immunization. The decay rate of the antibody titers showed surprisingly little effect of adjuvant, except for sustained-release polymer-based formulations. Adjuvants that gave high titers initially after boosting showed the greatest persistence of antibody titers (persistence defined as the residual titers at long times). These data show that high, long-lasting titers may be achieved by using sustained-release formulations, and these are likely the prime vaccine candidates for prophylaxis requiring prolonged sterilizing immunity.

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.

Adaptation to persistent growth in the H9 cell line renders a primary isolate of human immunodeficiency virus type 1 sensitive to neutralization by vaccine sera.

Seven diverse primary isolates of human immunodeficiency virus type 1 (HIV-1) were examined and found to be refractory to neutralization by antisera to recombinant gp120 (rgp120) protein from HIV-1 MN. This stands in marked contrast to the sensitivity exhibited by certain laboratory-adapted viruses. To understand the difference between primary and laboratory-adapted viruses, we adapted the primary virus ACH 168.10 to growth in the FDA/H9 cell line. ACH 168.10 was chosen because the V3 region of gp120 closely matches that of MN. After 4 weeks, infection became evident. The virus (168A) replicated in FDA/H9 cells with extensive cytopathic effect but was unchanged in sensitivity to antibody-mediated neutralization. Thus, growth in cell lines is not sufficient to render primary virus sensitive to neutralization. The 168A virus was, however, partially sensitive to CD4 immunoadhesin (CD4-Ig). Adaptation was continued to produce a persistently infected FDA/H9 culture that displayed minimal cytopathic effect. The virus (168C) was now sensitive to neutralization by MN rgp120 vaccine sera and by MN-specific monoclonal antibodies and showed increased sensitivity to HIVIG and CD4-Ig. 168C encoded three amino acid changes in gp120, including one within the V3 loop (I-166-->R, I-282-->N, G-318-->R). MN-specific monoclonal antibodies bound equally to the surface of cells infected with either neutralization-resistant or -sensitive virus. The coincidence of changes in neutralization sensitivity with changes in cell tropism and cytopathic effect suggests a common underlying mechanism(s) acting through the whole of the envelope protein complex.

Neutralizing antibody responses to autologous and heterologous isolates of human immunodeficiency virus.

Although laboratory-adapted strains of human immunodeficiency virus (HIV) are generally highly sensitive to neutralization by HIV-positive patient sera, we have found a more complex pattern of cross-neutralization and neutralization resistance among low-passage clinical isolates. These HIV isolates, like many other lentiviruses, resisted neutralization by the patient's own (autologous) antibodies. We assessed the degree of antigenic relatedness between different patient isolates of HIV through cross-neutralization with heterologous sera and virus isolates. Complicated patterns emerged, with variation in breadth of neutralization among individual plasmas and variation in frequency of neutralization among isolates. In longitudinal studies of individuals, we found that some but not all such patients develop a neutralizing response that "catches up" with their earlier isolates after a lag period. Taken together, these data suggest that an individual's immune response broadens with time because of cumulative exposure to multiple antigenic variants that arise throughout HIV disease.

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.