Development of a methodology for successful multigeneration life-cycle testing of the estuarine sheepshead minnow, Cyprinodon variegatus.

Evaluation of effects on fish reproduction and development during chemical exposures lasting for multiple generations is sometimes limited by variable reproductive responses and the time required for the exposure. Established testing methods and the short life cycle of the sheepshead minnow, Cyprinodon variegatus, make this species particularly suitable for use in identifying potential impacts of contaminants in estuarine and marine environments. This study describes the refinement of life-cycle exposure methods that increased the reliability of reproduction in sheepshead minnows and reduced the time to maturation for larvae and juvenile fishes. A test of three spawning chamber designs, three sex ratios, and two photoperiods identified conditions that reduced the coefficient of variation in egg production from >100% to as little as 32%. The most reliable results were produced with groups of three female and two male fishes (all of similar size) when they were placed in a rectangular chamber and acclimated for 12 days. A test water temperature of 26.5 +/- 2 degrees C and a 14L:10D photoperiod resulted in fish producing a mean of 74 embryos per female per day, with a coefficient of variation of 31.8%. Egg fertility exceeded 90%, with a hatch rate of 95% for normal embryos (>or=80% yolk) and a hatch rate of or=2.7 cm standard length) was critical for spawning readiness. Adult fish were prepared for the spawning assessment by adding frozen brine shrimp to their diet. Results of these experiments provide methods that are of particular interest in assessment of endocrine-disrupting chemicals that are known to affect reproduction.

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.

Development and qualification of a novel virus removal filter for cell culture applications.

Commercial bioreactors employing mammalian cell cultures to express biological or pharmaceutical products can become contaminated with adventitious viruses. The high expense of such a contamination can be reduced by passing all gases and fluids feeding the bioreactor through virus inactivation or removal steps, which act as viral barriers around the bioreactor. A novel virus barrier filter has been developed for removing viruses from serum-free cell culture media. This filter removes the 20 nm minute virus of mice by >3 log reduction value (LRV), the 28 nm bacteriophage PhiX174 by >4.5 LRV, the mycoplasma Acholeplasma laidlawii by > or =8.8 LRV, and the bacteria Brevundimonas diminuta by > or =9.2 LRV. Robust removal occurs primarily by size exclusion as demonstrated over a wide range of feedstocks and operating conditions. The filtered media are indistinguishable from unfiltered media in growth of cells to high densities, maintenance of cell viability, and productivity in expressing protein product. Insulin and transferrin show high passage through the filter. The virus barrier filter can be autoclaved. The relatively high membrane permeability enables the use of a moderate filtration area.

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.

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.

Assessment of human immunodeficiency virus expression in cocultures of peripheral blood mononuclear cells from healthy seropositive subjects.

We have evaluated a number of methodological variables effecting the expression of human immunodeficiency virus (HIV) in cultured peripheral blood mononuclear cells (PBMCs) of 93 healthy anti-HIV-positive and 72 healthy seronegative subjects. For optimal HIV recovery, PBMCs had to be freshly separated from whole blood. Short-term freezing of purified PBMCs was practically advantageous and actually resulted in more rapid virus recovery. The minimal number of PBMCs necessary for virus expression was determined by dilutional cultures and varied from 10(2) to 10(6) cells. HIV expression was demonstrated initially at the cellular level by immunocytochemical detection of HIV core and envelope proteins using a mixture of monoclonal antibodies, subsequently confirmed by detection of viral antigens and reverse transcriptase (RT) in the culture supernatants. HIV recovery was not improved following induction with 5-iodo-2'-deoxyuridine (IUDR) and only marginally improved following depletion of the CD8+-suppressor cell population in the PBMC specimens. The overall frequency of HIV detection in cultures was 84% in healthy seropositive subjects, whereas none of the PBMCs from 72 seronegative persons yielded HIV expression.