IAVI Report - February / March 2001
Patricia Kahn

Merck's DNA- and Adenovirus-based Vaccine
In two much-anticipated talks, scientists from Merck Research Labs gave the first public presentation on the company's current HIV vaccine program. Merck's entry into the field is widely seen as an important boost, given how few large pharmaceutical companies are involved. (Another newcomer is GlaxoSmithKline; see Vaccine Briefs).

The Merck researchers described some of the extensive pre-clinical studies that led the company to move two types of vaccines-those based on naked DNA and on adenovirus vector-into Phase I trials. Both vaccines target cellular immune responses only, although Merck is actively searching for approaches that stimulate strong neutralizing antibodies, according to Emilio Emini, vice president of antiviral and vaccine research. Merck's aim is to develop candidates both as preventive and therapeutic vaccines.

John Shiver, Director of Vaccine Research, began by showing comparative studies of single types of vaccines (i.e., no prime-boost combinations) in rhesus macaques. These were: plasmid DNA (5mg) in saline, alum or CRL1005 (an ionic block co-polymer adjuvant); and MVA- (modified vaccinia Ankara) and adenovirus type 5 (Ad5)-based viral vectors, both used in the "highest achievable" doses. The Ad5-vector cannot replicate or integrate into the host genome, and its encoded proteins persist only for a "finite" time, according to Emini. All vaccines contained an SIV gag gene (optimized for human codon usage) but no env, and were administered intramuscularly either three or four times (for viral vectors or DNA, respectively) over 32 weeks to three animals each. Three months after the last immunization, all vaccinated animals and six controls were challenged i.v. with a high dose (50 m.i.d.) of SHIV89.6P.

Prior to challenge, animals given Ad-5 showed the highest immune responses, followed closely by DNA/ CRL1005 adjuvant (as measured by intracellular cytokine staining and ELISPOT for interferon-gamma production, and by tetramer binding). Responses to DNA in saline or alum were much lower, showing a clear advantage of adjuvanting the DNA with CRL1005. CD4 T-cell responses were also induced in most vaccinees and tended to predominate in the DNA group, while Ad5 vaccinees had significantly higher CD8 cell responses. 

At 180 days post-challenge, all vaccinated animals were alive and apparently healthy (although infected), compared with 4/6 controls that had died and a fifth that was symptomatic. However, there were clear differences among the groups. The Ad5-vaccinated monkeys showed the best clinical course: CD4 counts remained high, peak viremia was blunted by 1.5 logs and viral load gradually brought under control. Animals given DNA/CRL1005 adjuvant were next best, followed by MVA, both initially showing CD4 cell decline but then recovering and successfully controlling viral load. DNA in alum/MPL or saline led to the biggest drop in CD4 cells and the least control of viremia.

Speaking two days later, Emilio Emini presented immunogenicity data from DNA or Ad5 vaccines with HIV gag (rather than SIV). Animals given DNA/CRL1005 adjuvant and boosted with Ad5 showed a 5-10-fold increase in the number of HIV-specific T-cells (by ELISPOT) after the boost. That generally reflected both CD4 and CD8 T-cell responses, again with CD8 T-cells predominating after the Ad5 boost (even in animals with predominantly CD4 responses following the prime). Unadjuvanted DNA was a less effective prime, with boosted monkeys showing little increase by ELISPOT. 

Emini also addressed the issue of pre-existing immunity to adenovirus, a key to the feasibility of using this vector as a vaccine since about 10% of the U.S. population has significant neutralizing antibodies to adenovirus and another 40% shows lower levels, according to Emini. In these studies, 6 monkeys were pre-exposed to adenovirus and immunized later with a high dose (10¹¹ particles) of Ad5-gag. While the presence of neutralizing antibodies reduced the strength of the T-cell responses several fold, it did not eliminate them, leading Emini to express optimism that pre-existing immunity could be overcome, especially with a DNA prime/Ad5 boost strategy. As support, he cited the effectiveness of the Ad5-gag boost described above (which used 10⁷ particles), suggesting that a 10⁹ particle dose, even if neutralized by 99%, should still induce good responses.

Lastly, Emini reported that PBMCs from about two dozen people infected with HIV subtype B gave "comparable" responses (in ELISPOT assays) against peptides from Gag and Nef consensus sequences of subtypes B and C, lending some support to the feasibility of broadly reactive vaccines (at least those targeting cellular immunity). He also said that nef and pol will be added to Merck's vaccines alongside gag, based on the company's data showing that a high proportion of HIV-infected people with good immune function and low but detectable viral load respond to these proteins. 

The company has several Phase I studies underway. A DNA-gag trial in uninfected people began last year, and one in infected people a few weeks ago. Ad5-gag studies recently started in uninfected volunteers and will soon begin in infected people. Depending on these results, some recipients of the DNA vaccine may be boosted with adenovirus.

Protection with Adeno-Associated Virus Vectors
Phil Johnson (Children's Research Institute, Columbus, Ohio) showed macaque data from ongoing studies of an SIV vaccine based on vectors of adeno-associated virus (AAV), a single-stranded DNA virus common in the population. The vaccine is a mixture of 3 types of AAV particles that collectively carry SIV env, rev, gag, int and protease. In 8 immunized animals challenged i.v. with a high dose (5-50 m.i.d.) of SIV-E660, a strain that is difficult to protect against, viral peak was reduced by about 1.3 logs, setpoint by 1.7 logs, and viral load by 3 logs. Vaccinated animals were still healthy at 6 months, while 3/8 controls died. All vaccinees showed both CTL responses and neutralizing antibodies, which persisted for at least 14 months. Viral load after challenge correlated with both neutralizing antibody titer on the day of challenge and peak levels of SIV-specific CD8 cells. In a prime-boost study of the AAV vaccine with SIV-DNA, peak viremia was blunted even more (by 2.5 logs).

Johnson also reported that the vector, which targets quiescent cells, shows extreme longevity (>17 months) in the muscle cells of immunized monkeys. Unlike the wild-type parental virus, it appears to persist in host cells as an episome rather than integrated into chromosomal DNA. Together with Targeted Genetics (Seattle), Johnson's group is making a clade C vaccine from South African isolates, under an IAVI-sponsored collaboration.

Sabin Polio Vaccine as an HIV Vector
Shane Crotty (University of California at San Francisco) presented a macaque study of SIV vaccines made with vectors of the live Sabin vaccine, known to induce potent, long-lasting immunity when taken orally. Since the vector can fit only very small pieces of foreign DNA (500-700 bases), the researchers used two mixtures of 20 different recombinant viruses representing the entire SIV genome in overlapping fragments. Seven animals were immunized intranasally four times and challenged vaginally nine weeks later with the highly virulent SIVmac251. While 12 unvaccinated controls became infected and 3 of 6 closely monitored controls died by week 40 (and the other three showed disease symptoms), two vaccinees had no detectable viremia; two were able to control viremia, and the remaining three became highly viremic, like the controls. All 7 vaccinated monkeys maintained body weight, unlike controls. The HIV-polio virions are also being tested as boosts in a prime-boost regimen. 

Crotty says that these viruses are 1,000-10,000 more infectious in humans than in macaques, and therefore monkeys may not be a good model for these vectors. His group, headed by Raul Andino, is working on human trials of the approach. 

Immune Responses in Oxford Phase I Trial 
In a talk on cellular immune responses to HIV, Andrew McMichael of Oxford University presented some preliminary results from the UK's ongoing HIV-DNA vaccine trial. (A second Phase I trial recently began in Nairobi.) The vaccine, designed by Oxford's Tomas Hanke and manufactured by Cobra Ltd., carries most of the gag gene (containing many known CD8 and some CD4 epitopes) from subtype A and a string of CTL epitopes from pol, nef and env. The 18 vaccinated volunteers received either 100ug or 500ug DNA (low doses chosen for initial human safety studies)

Four weeks after the second immunization, 9/18 volunteers showed positive responses in ELISPOT assays of CD8 cells expressing interferon-gamma (performed by Matilu Mwau). The data have not yet been unblinded in terms of the two dosage groups. Plans are to use this DNA vaccine with an MVA boost, which just entered separate Phase I trials in Oxford (see Vaccine Briefs). •

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