The United Nations estimates that over 40 million people are currently infected with HIV-1. Each day approximately 16 000 new infections and half as many deaths occur as a result of AIDS. One of the most substantial challenges to the design of an effective global HIV-1 vaccine is virus variability. As a result of an error-prone reverse transcriptase and lack of polymerase-related proof-reading function, heterogeneity occurs throughout the HIV-1 viral genome. Mutations affect all viral proteins including envelope, the target of neutralizing antibody . Our current goal is to address HIV-1 diversity by the design and testing of a multienvelope vaccine that includes recombinant DNA, vaccinia virus, and protein vectors. As an initial step in this process, we have produced a purified recombinant envelope protein of subtype D and entered this vaccine into a phase I clinical trial at St Jude Children's Research Hospital (SJCRH) in Memphis, Tennessee, USA.
To produce EnvPro, recombinant Chinese hamster ovary cells expressing UG92005 gp140 envelope gene (GenBank accession no. AF338704) were grown under current good manufacturing practice conditions for the collection of supernatants and isolation of soluble protein. Envelope was purified by affinity chromatography and formulated with alum (Reheis, Berkeley Heights, New Jersey, USA). Single vaccine doses comprised 100 μg envelope protein mixed with 500 μg alum and were administered by the intramuscular route.
This study was designed as a single-center trial employing standard criteria (as defined in the HVTN Table for Grading Severity of Adverse Experiences, 18 September 2002), to evaluate the tolerability and safety of intramuscular EnvPro. The study was approved by the internal regulatory committees of SJCRH (including the Institutional Review Board) and an investigational new drug application was approved by the United States Food and Drug Administration. Signed informed consent was received from all study participants. The study enrolled nine participants (four women and five men, average age 38.2 years) and evaluated each for vaccine tolerability, focusing particularly on the 28 days after vaccination. Participants were enrolled serially, one every 28 days (as requested by the SJCRH Institutional Review Board), such that only one participant was being observed for tolerability and safety at any given time. Two doses of vaccine were administered 8 weeks apart. This dosing schedule was based on our preclinical studies and on studies of other purified protein vaccines .
Inclusion criteria required study participants to be healthy adults (> 18 years) and HIV-1 negative with normal complete blood counts, CD4 T-cell counts (≥ 400 cells/μl), aspartate aminotransferase, alanine aminotransferase, serum creatinine, sodium, potassium and chloride and creatine phosphokinase. Prospective volunteers were excluded if they had a history of immunosuppressive illness, chronic illness, or the use of any immunosuppressive medications, had received live attenuated vaccines within 60 days of study enrollment, had received experimental agents within 30 days of study enrollment, had received blood products or immunoglobulin in the past 6 months, were allergic to gentamicin, or were pregnant or nursing at the time of vaccination.
All candidates enrolled in this study received counselling for safe sexual practices. Recruitment and compliance were excellent.
As demonstrated in Fig. 1(a), EnvPro was uniformly well tolerated and participants did not develop laboratory abnormalities. Adverse events that were considered possibly, probably or definitely vaccine related were grade 1 only and included pain, warmth or swelling at the injection sight, headache, fatigue, malaise, myalgia, and nausea. No unexpected vaccine-related adverse events were noted. Adverse events were largely related to the intramuscular inoculation itself (e.g. tenderness, mild swelling or perceived warmth at the site of inoculation) or systemic manifestations such as general malaise, headache, fatigue or nausea. Four of the nine study participants reported no adverse events considered to be related to vaccination.
Immune responses were examined using the Abbott clinical enzyme-linked immunosorbent assay [HIVAB HIV-1/HIV-2 (recombinant DNA) enzyme immunoassay; Abbott Laboratories, Abbott Park, Illinois, USA]. Results are shown for each of the nine volunteers in Fig. 1(b). Each volunteer exhibited a classic immune response in that little activity was seen after the priming immunization, but assays scored positively for all volunteers after the booster immunization. This frequency of positive antibody responses in a standard serological test (100%) exceeded that observed in several other studies of HIV-1 protein vaccines , highlighting the potency of the gp140/alum formulation. In several participants responses waned, but in two volunteers (participants 509 and 511), responses remained positive for at least 3 years postimmunization and for a third volunteer (participant 513) responses remained positive for at least 2 years (this participant has not yet completed the 3-year study, data not shown). We note that the envelope antigen in the Abbott assay is of subtype B, whereas the EnvPro vaccine is subtype D derived. Results illustrate that HIV-1 subtypes and immunotypes are not the same [4,5].
As expected based on preclinical studies, EnvPro vaccinations did not have the potency of prime-boost multivectored regimens [6,7]; neutralizing antibody or T-cell responses of high magnitude were not identified (data not shown).
Future plans are to combine EnvPro with DNA and vaccinia virus recombinant vectors in a prime-boost clinical protocol [6,8–10]. As with EnvPro, our DNA and vaccinia virus vectors  have also been tested in individual phase I safety trials and elicited no unexpected vaccine-related adverse events. Our preclinical studies indicate that the prime-boost regimen will elicit responses of greater magnitude, breadth and durability.
In conclusion, we describe the safety and immunogenicity of the first subtype D HIV-1 gp140 envelope protein vaccine to be tested in a phase I clinical trial. These results are promising and encourage the advancement of EnvPro to a trial that combines multiple vectors and multiple envelopes. A clinical trial with a multienvelope vaccine (utilizing recombinant DNA, vaccinia virus and purified protein; D–V–P) has now been approved by the United States Food and Drug Administration. Results from the study described in this report suggest that virtually every volunteer will respond to the D–V–P combination vaccine. On the basis of results from small and large animal studies , broad, durable and robust B and T-cell immune activities are expected when EnvPro is tested as part of this multivector, multienvelope vaccine study in humans.
The authors would like to thank Dr James Bradac and the World Health Organization for assisting in the acquisition of the UG92005 virus, from which the EnvPro gp140 sequence was derived. They also thank Drs James Mullins and Harriet Robinson for the pJW4303 vector used in the process of recombinant Chinese hamster ovary cell preparation; C. Sinnock for assistance with the clinical trial; J. Knight, and R. Lake for assistance with quality assurance for good manufacturing practice production and regulatory affairs; and Randall Hayden and the Pathology Department for the conduct of Abbott assays. The authors are also grateful to the volunteers without whom the study would not have been possible.
Sponsorship: This work was partly funded by National Institutes of Health NIAID grant P01 AI45142, Federated Department Stores, the Mitchell Fund, the Anderson Foundation, the Pendleton Fund, the Pioneer Fund and the American Lebanese Syrian Associated Charities.
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