The STEP trial

Promising signs

Hopes were high that the STEP trial would produce a significant positive result. The size of the trial had been doubled in August 2005 after CD8 responses seen in volunteers appeared promisingly high and also relatively independent of the degree of pre-existing immunity to the adenovirus 5 vector. This latter consideration was particularly heartening as it had been thought that pre-existing immunity to the vector would be the biggest limiting factor on the success of the vaccine (Isaacs 2004). The trial protocol was therefore relaxed to take in a second wave of recruits with higher titres of anti-ad5 antibodies.

Aidsmap, reporting from the AIDS Vaccine Conference in Amsterdam in September 2006, commented that “the gloom that has dominated the AIDS vaccine field since the pessimistic AIDS Vaccine ’04 conference in Lausanne began to lift further last week with the confident statement by a leading researcher that 2006 has been ‘a vintage year for vaccine development.’ Why are vaccine researchers beginning to feel more optimistic?”

The first reason was the impressive immune responses, or immunogenicity, demonstrated by at least three adenovirus-based HIV vaccines presented at the meeting.

The HIV Vaccine Trials Network (HVTN) 054 study (Peiperl 2006) tested what dose of adenovirus 5 might be needed in order to produce an immunogenic response, and also tested the safety of different adenovirus doses. Since up to 90% of the adult population may have some level of neutralising antibody to adenoviruses in sub-Saharan Africa, the HIV Vaccine Trials Network wanted to test whether it was necessary, or safe, to use a very high dose of adenovirus.

Higher doses caused flu-like effects without any increase in the ELISPOT interferon-gamma responses that are used to measure HIV immunogenicity.  Eighty-seven per cent of the lower dose group showed positive ELISPOT reactions for any HIV antigen included in the vaccine by day 28, although the proportion who registered reactions to Gag, Pol and at least one Env gene was not reported. A similar proportion (85%) showed immunogenicity in the higher dose arm, leading the investigators to conclude that the lower dose should be taken forward.

Analysis of Phase I participants who received the Merck adenovirus-based ad5 vaccine presented at the Vaccines 06 meeting (Casimiro 2006) showed that around 70% of participants who produced CD8 T-cell responses to HIV-1 subtype B also had T-cell responses to consensus sequences of subtype A and C as measured by ELISPOT interferon-gamma assay, although the proportion of responders to subtype B nef that were responsive to subtypes A or C nef was much lower, at less than 30%.

A number of presenters at the meeting emphasised the need to look for the most highly conserved sequences of the HIV-1 group M, to which all HIV-1 clades belong, in order to identify the antigens that could be targeted by a vaccine. Bette Korber of Los Alamos National Laboratory in the United States said that as recombinant viruses spread, it made sense to target the similarities in group M rather than developing vaccines for each different subtype. “There’s no way we’ll be able to cope otherwise,” she said.

She explained that the search for conserved sites and critical differences between viruses need not be as difficult was once thought.

“In conserved HIV proteins, the positions where substitutions are tolerated usually oscillate between a few common substitutions,” she said. “The observed frequencies are the result of a balance between fitness, immune escape and the evolutionary relationships of the strains we sample.”

In HIV-1 subtype B for example, her research group has found that within pol, the most highly conserved HIV antigen, only four of 1004 nucleotide positions within the genome require as many as four amino acid changes, a finding which reduces the number of potential variations that need to be covered by a potential vaccine.

Prof. Andrew McMichael’s research group at the University of Oxford is also looking at constructing a vaccine which is immunogenic across subtypes, and reported at the meeting that a construct called HIVCON spanning the most conserved regions of subtypes A, B, C and D has proven highly immunogenic when tested against plasma from healthy volunteers and HIV-positive people (Letorneau 2006).

So, after years of fruitless attempts to develop vaccines that stimulated broadly protective neutralising antibodies which prevent HIV infection, vaccine researchers had begun to concentrate on vaccines that would stimulate T-cell responses – and thought they were seeing some very promising results.

“These promising results suggest that the success of the Merck adenovirus-based HIV vaccine is not a flash in the pan, and that the strategy of using a strongly immunogenic vector to stimulate T-cell responses is working,” commented the Aidsmap report. “What’s less clear is what the end result of vaccination will be: prevention of infection or control of HIV disease progression? We should know by 2008 or 2009 whether this vaccine has some protective effect against HIV transmission, at which point it could move into much larger Phase III trials required for licensing.”

The conference was also excited by research showing that it was now possible to measure the quality, or `polyfunctionality` of the immune responses induced by T-cell vaccines in a much more comprehensive way. Viral control, whether in animals or in HIV-positive humans, depended on a broad array of T-cell functions being maintained in good working order.

However, in a warning that was remembered a year later, Jerald Sadoff of the Aereas Global TB Vaccine Foundation told the conference that determining which immune responses corresponded with true immune protection was still a puzzle for researchers. While correlates of protection help a lot in proving that a vaccine works, he said, “often you don’t have your correlate until you prove it works – you assume it works until you prove it.”

Until now, the only factor found in common among people who control HIV after infection has been strong T-cell responses to HIV Gag proteins.

In contrast, people with strong responses to HIV envelope proteins tended to have higher viral load levels. This may be because immune responses to envelope proteins drove the virus to diversify its coat to escape immune detection much more quickly.

This finding, together with the recent discovery that envelope proteins – specifically HIV gp120 – shift the immune system response away from vigorous HIV-specific T-cell responses, have led some researchers to question whether envelope proteins should be included in vaccines at all, according to Dr Jill Gilmour of the International AIDS Vaccine Initiative.

Professor John Moore of Cornell University agrees that the HIV envelope remained a major problem in vaccine design. “The responses are essentially fiendish. We have to make a global influenza vaccine every year to respond to variation; in comparison, HIV envelope variation is vastly greater.”

Yet there are human neutralising antibodies that can bind to HIV envelope spikes, and the International AIDS Vaccine Initiative was funding a research consortium to search more intensively for HIV antigens that generated neutralising antibodies. The most conserved sites, however, were well hidden and much less frequently displayed to the immune system, making it very difficult to apply neutralising antibodies to these sites.

“Whether it will be necessary for a successful AIDS vaccine to induce a healthy neutralising antibody response alongside a T-cell response remains to be seen,” said Aidsmap.

The other reason for optimism was the development of a humanised mouse model for vaccine testing, reported at the conference by a team from the University of Texas Southwestern Medical Center. Animal modelling of HIV infection is limited by the fact that HIV is a human immunodeficiency virus, and primates are not susceptible to infection by it, so primate experiments must use a genetically engineered simian immunodeficiency virus in order to deliver HIV genes, commonly called SHIV. The University of Texas group had at last succeeded in transplanting human CD34+ stem cells into the haematopoietic (blood-cell-making) systems of immunodeficient mice that had already received transplants of human liver and thymic tissue, leading to an immune reconstitution with human lymphocytes. HIV infection of the mice resulted in a predictable pattern of disease progression, and the mouse model, said vaccine researchers, had the potential to allow much faster animal testing of vaccine candidates.

Closure of the STEP trial

It was with a sense of shock, then, that the HIV vaccine community heard on 21 September 2007 that trials of the most promising HIV vaccine to date had been halted following news that the vaccine did not protect against HIV infection.

Vaccination in the STEP trial was being discontinued because the vaccine was not effective, an announcement by the co-sponsors of this clinical trial, Merck & Co., Inc. and the HIV Vaccine Trials Network (HVTN), said. HVTN is funded by the National Institute of Allergy and Infectious Diseases (NIAID), part of the US National Institutes of Health.

The independent Data Safety Monitoring Board (DSMB) for STEP had reviewed safety data and results of an interim efficacy analysis of the study, and recommended that vaccination be discontinued because the STEP trial would not meet its efficacy endpoints.

Study investigators were instructed to discontinue vaccinating volunteers in this study and to monitor them in accordance with the study protocol. Enrolment and vaccination in the Phambili trial, and two additional Phase I trials, were also being discontinued. The DSMB for the Phambili trial would evaluate the available data.

STEP evaluated two primary efficacy endpoints: whether the vaccine prevented HIV infection and whether the vaccine reduced the amount of virus in those who developed infection. As planned, an interim efficacy analysis was conducted in the approximately 1500 volunteers expected to have the best response to the vaccine because they had low levels of pre-existing immunity to adenovirus 5.

The vaccine did not prevent infection: in volunteers who received at least one dose of the three-dose vaccine series, 24 cases of HIV infection were observed in the 741 volunteers who received vaccine and 21 cases of HIV infection were observed in the 762 participants in the placebo group.

In the subgroup who had received at least two vaccinations and who were HIV-negative for at least the first twelve weeks of the trial, 19 cases of HIV infection were observed in the 672 volunteers who received vaccine and eleven cases were observed in the 691 volunteers who received placebo.

These were interim figures: later on, when all figures were collected, it was found that there were 49 infections in patients receiving the vaccine and 33 in those receiving placebo. This 48% higher rate of infection in vaccine recipients was not statistically significant and at this point it was unclear if it had a cause.

In addition, the vaccine did not reduce the amount of virus in the bloodstream of those who became infected; HIV RNA levels approximately eight to twelve weeks after diagnosis of infection were similar in the vaccine and the placebo arms. The geometric means of the HIV RNA levels in the blood of infected individuals, the standard measure of ongoing HIV replication, were approximately 40,000 copies/ml in the vaccine group and approximately 37,000 copies/ml in the placebo group. Additional analyses will be conducted on the entire study population and will be shared with the scientific community.

Study volunteers were followed for approximately thirteen months. Overall adverse event rates were generally similar among the two groups, except for a higher rate of local injection-site related reactions in the vaccine group.

"This trial was the first test of concept trial that provided us information on this vaccine more quickly and efficiently than with a traditional Phase III design. While we are very disappointed that this vaccine candidate did not demonstrate protection, the data from this trial will provide critical insights into this disease and future vaccine development," said Larry Corey, M.D., principal investigator of the HVTN.

"This is a huge disappointment for all of us who have been involved in the search for an HIV vaccine," said Glenda Gray, M.D., principal investigator of the HVTN sponsored Phambili trial. "HIV is ravaging our communities, and all the scientists, participants and communities involved in HIV vaccine studies have been affected by this epidemic. The scientific community must continue the race to find a vaccine to help secure an HIV free generation for the future."

It would be difficult to exaggerate the sense of disappointment felt about STEP. With the earlier AIDSVAX envelope-protein vaccine, there had been plenty of sceptics around before the trial who doubted whether it would have any efficacy. Hopes had been high, however, that the STEP vaccine, even if it did not reduce HIV incidence, would at least reduce viral loads in those infected, as it had done in animal studies. The finding that there was no difference whatsoever in the viral loads of seroconverters who took vaccine against those who took placebo was not only disappointing, it was an indication that some of the assumptions and methodology behind the trial and indeed all trials of CD8-stimulating vaccines might need revising. It had been hoped that at the very least the CD8 response created by the vaccine – a real and measurable effect that had already been seen in previous studies – would moderate the course of infection. The fact that it did not do so has caused researchers to ask, in the words of HVTN’s Judy McElrath, “Was the concept of a T-cell-based HIV-1 vaccine clinically not valid, or is the lack of efficacy specific to the Ad5 vaccine candidate?”

Did the adenovirus-5 vaccine make some people more vulnerable to HIV?

Worse was to come, however. A hastily-conducted post-hoc analysis of the trial was done, which reported at the HVTN Conference in Seattle on 7 November 2007 (HVTN 2007). This uncovered a more alarming possibility: the vaccine, though it could not possibly have caused HIV infection in itself, may have made some participants more vulnerable to HIV.

A round table of experts convened at the conference heard that the higher rates of infection in vaccine recipients were not statistically significant: in other words, they could be a random fluctuation due to chance alone. But the higher rates of HIV infection in at least some of the volunteers given the vaccine are nonetheless more likely to be real than random, the analysis suggests.

In the STEP trial 1500 patients at high risk of HIV in the Americas and Australia were originally recruited, starting in December 2004. They were largely gay men and female sex workers. Sixty-two per cent were male and the average age was 29.

This was a population with high levels of risk for HIV acquisition through sex. Half of the women and 21% of the men had had at least 20 sex partners in the previous six months; 75% of the women and nearly 60% of the men had had unprotected anal or vaginal sex in the same period; and one in five of the women and one in three of the men had had sex with someone whose HIV status they did not know.

There was only one infection in female volunteers in STEP. While this looks like a startling finding at first, it has not been highlighted in the analysis, and reflects three facts; firstly, women lagged behind men in trial recruitment and did not have so much time to catch HIV; secondly, HIV prevalence amongst heterosexual male contacts of female trial volunteers was considerably lower than it was in the partners of gay male volunteers; thirdly, it illustrates that anal sex transmits HIV more easily. The HIV infection figures below are of the men in the study and exclude the one woman who became infected.

The original news released on September 21st, then, was already disappointing, but the post-hoc analysis brought worse news. An apparently slight excess of HIV infection in those receiving vaccine started looking more significant when the results were stratified according to the level of pre-existing immunity volunteers had to the Ad5 virus.

Originally, people with high levels of pre-existing immunity to the Ad5 adenovirus had been excluded from the STEP trial, but a decision to double the trial’s size had been taken in August 2005 when reasonable immune responses were observed in this group. The trial had reached its target of 3000 volunteers by March 2007, and the analyses that follow are based on 1503 volunteers who received at least one shot of vaccine or placebo and 1363 volunteers who received at least two shots (the full schedule was three injections at zero, one and six months).

Volunteers were stratified into four groups according to ad5 antibody levels in their blood:

• In those with the lowest antibody levels, 4% of volunteers caught HIV regardless of whether they had vaccine or placebo.

• In those with medium-low levels, 4.4% of those given vaccine caught HIV but only 2.2% given placebo, and in those with medium-high levels, 6.1% given vaccine caught HIV and 3.0% given placebo, so twice as many given the vaccine caught HIV in both of these groups.

• In those with the highest levels of ad5 antibodies, 4.4% given vaccine versus 1.2% given placebo became infected - 3.5 times as many, though in this case numbers were very small (seven vaccinees and two on placebo).

In all cases the confidence intervals overlapped, and no result in any category was near statistical significance. But the direction of change was consistent – and alarming.

The classical definition of ‘statistical significance’ is that there is a less than one-in-20 probability that the effects observed could be due to chance. The round table heard that there was a one-in-17 probability that the higher rates of HIV infection seen in male volunteers who had high levels of pre-existing ad5 immunity could have been a chance effect, as opposed to an effect of the vaccine. In this group, 21 volunteers given vaccine acquired HIV versus nine given placebo. Over the volunteer group as a whole the chance of this happening was one in 6.5.

Another possibility to be considered is that high ad5 immunity conferred some kind of pre-existing resistance to HIV infection in the placebo group – which was abrogated by the vaccine. If this is taken into account it means that there is some characteristic in both the vaccine and the placebo groups that may have to be taken into account, and this then creates what is called a “double sided” possibility- meaning that the probability that observed effects are due to chance is halved.

This meant that there was then one chance in 13 that the difference in infection rates observed in those with high ad5 immunity was a random effect, and only one in 35 if only the men are considered – within the boundary of statistical significance.

Steve Self of HVTN said that the classical statistical significance test was useful when testing to see if something worked. But when evaluating a possibly harmful effect, “probability values much smaller than the usual threshold values still provide some indication of strength of evidence.”

Saying that chance is the explanation for differences in infection rates, he added, was only an “explanation of exclusion” – in other words, it excludes other variables that researchers can measure but does not exclude ones they cannot measure or have not thought of.

Keith Gottesdiener of Merck added: "This difference is clinically important for at least one subgroup, the high Ad5. I don't really need any statistics to make a declaration that it's an important factor to take into consideration."

What differences were there between the volunteer groups? There were none between the vaccine and placebo groups: this was a very well-randomised trial. But there were significant differences between those with low and high levels of ad5 immunity. People with higher levels of ad5 immunity, recruited in the second wave of enrolment, were twice as likely to be female, nearly half as likely to be white and gay, and somewhat younger. We know there is already a gender bias in the infections, but was this due to gender, because more women were black, or for some other reason?

One thing can be ruled out: there were no behavioural differences between those receiving placebo and those receiving vaccine. As this was a double-blinded trial, those receiving vaccine (or those administering it) would have to guess correctly what they had received, and then change their behaviour accordingly. Susan Buchbinder of the University of California in San Francisco said there was no evidence of either of these things happening.

In terms of behavioural differences between those with low and high levels of pre-existing ad5 immunity, the few differences there were, such as lower levels of unprotected anal sex amongst men with high ad5 immunity, might have been expected to protect them from HIV, not expose them to it.

The men with high pre-existing immunity to ad5 were also half as likely to be circumcised. This was a potentially significant finding. Nearly two-thirds of the men in the trial (64%) were circumcised. Excluding men with uncertain circumcision status, there were 26 infections each in circumcised vaccine and placebo recipients, but in uncircumcised recipients only six receiving placebo caught HIV versus 26 receiving vaccine – the latter figure representing a fourfold greater risk of HIV acquisition for vaccine over placebo, and a 56% greater risk for uncircumcised over circumcised. The lower figure in men receiving placebo is also intriguing.

Were there any differences in immune response between vaccinees who contracted HIV and ones who did not? There is a suggestion this may be the case.

As expected, volunteers with high levels of pre-existing ad5 immunity had lower immune responses to the HIV vaccine: about 72% of low-immunity volunteers and 54% of high-immunity volunteers had a significant immune response to any one of the three HIV proteins.

But in high-immunity patients who caught HIV, only 43% had an immune response. However it is difficult to see any consistent pattern in these immune responses – the inter-individual variability is far higher than that between groups and again, no one difference has statistical significance.

Another possibility being looked at is that the immune responses generated to HIV were too narrow to deal with most viruses. Follow-up studies will look in much more detail at the immune responses generated in vaccine recipients and why they seemed to have no effect on HIV.

Could the vaccine have stimulated the immune system in an unhelpful way – one that sensitised, rather than immunised, people to HIV? If this turns out to be the case, it would be of great concern, because it could mean that recipients of the V520 vaccine could be super-receptive to HIV for a long time – possibly for the rest of their lives. Immune responses to the vaccine do appear to be long-lasting and had faded only slightly in a group of early vaccinees two years after injection.

It is for this reason that there is some urgency directed to the process of ‘unblinding’ the STEP trial – informing participants whether they in fact received the vaccine or the placebo. This is not easy to do in a hurry as participant records are anonymised precisely to avoid accidental or unauthorised unblinding.

Glenda Gray, Phambili’s national Lead Investigator in South Africa, described the process of informing the 800 volunteers in the Phambili trial at the HVTN meeting – a process that took two weeks to complete. Initially some of the STEP investigators wanted to keep their trial blinded to get ‘clean’ data but after several days of debate, a decision to unblind the trial was taken.

The STEP trial failure concluded a year of disappointing results in prevention trials, with several other trials, including ones of the female diaphragm and of the microbicide cellulose sulphate, being stopped due to lack of efficacy. In the CONRAD cellulose sulphate trial there were also more infections in the intervention than in the control arm of the trial, though again this was not statistically significant.

“The STEP Trial is not the first one that has shown this trend,” Mitchell Warren of the AIDS Vaccine Advocacy Coalition told Aidsmap. “These are figures that trend in an unpleasant direction.”

He agreed that it would be a good idea to take stock and press the pause button on forthcoming efficacy trials of prevention technologies until better correlates of protection had been found. “We need to create a very high standard before investing the kind of resources we need in another efficacy trial. I don’t know how high that bar should be: but we need to put one there.”

At the same time he does not believe that the STEP trial in itself should be seen as a failure. “It essentially took three years to get an answer for a vaccine efficacy trial, which is fast for one this size, and is a tribute to its design,” he says. “We got an answer. It was one we don’t like, but it was an answer.”

The role of circumcision

More information on the STEP trial emerged at the CROI conference in February 2008 (Robertson 2008). Further data were presented suggesting that the ad5 vaccine specifically increased the vulnerability of uncircumcised men to infection through insertive anal sex.

Over the whole trial group, the difference in infection rates between vaccine and placebo recipients was only marginally statistically significant, with a probability of 0.044 if the effect observed was caused by the vaccine alone (so-called ‘one-tailed’ probability), and 0.077 if the effect was caused by some combination of a direct vaccine effect and some kind of protective characteristic of the placebo group that was removed (‘two-tailed’ probability).

In the group that had high levels of adenovirus immunity, however, there were 21 infections in vaccine recipients and only nine in placebo recipients, and this was statistically significant (p = 0.02 one-tailed, 0.029 two-tailed).

Merck’s Mark Robertson, presenting preliminary data on immune responses in the trial (Robertson 2008), showed that volunteers with high ad5 immunity levels had higher levels of generalised CD4 cell activation. This difference was even more marked in HIV seroconverters than those who remained uninfected, so some kind of inflammatory interaction with the vaccine vector may be to blame.

But presenter Susan Buchbinder of UCSF said that the latter possibility appeared stronger. The incidence of HIV infection, at about 4-5% a year, was the same in vaccine recipients regardless of their ad5 immunity level. However the incidence of infection was lower in placebo recipients who had higher levels of ad5 immunity, ranging from 4% a year in those with the lowest level to 1.2% a year in those with the highest level. The vaccine may therefore somehow have removed a protective effect of ad5 immunity.

Both in a univariate model and in a multivariate model that progressively eliminated other possible confounding factors, high ad5 immunity conferred a threefold higher risk of infection in vaccine recipients than in placebo recipients, compared to no increased risk in people with low ad5 immunity when compared to the placebo group.

However, Buchbinder added, the real reason for the additional risk in vaccine recipients may have had little to do with ad5 immunity, or it may only have had an accessory role.

There was one more important risk factor for HIV acquisition in vaccine recipients when compared to the placebo group: circumcision, or rather, the lack of it.

Uncircumcised vaccine recipients were, in univariate and multivariate analyses, four times more likely to become infected with HIV if they received vaccine than if they received placebo. In contrast there was no difference at all in infection rates between circumcised vaccine versus placebo recipients.

In questions after the presentation, Buchbinder commented that the risk to uncircumcised men was greater than the risk to men with high ad5 immunity, and that the latter may be a passive marker for the former. This was because the men with high ad5 immunity, who were largely recruited in the second wave, also tended to come from countries and communities with lower rates of circumcision; they were younger and more likely to come from Latin America, for instance.

She also commented that preliminary data hinted that the enhancement of infection risk was specifically seen in uncircumcised gay men who largely or exclusively had insertive anal sex. This would imply that the vaccine was abrogating some immune mechanism that normally protected uncircumcised ‘tops’ from infection through the mucosa of the foreskin.

However, this would not explain in itself why placebo recipients were less likely to get HIV if they had high ad5 immunity, so the two effects may be synergistic.

There are still many questions left unanswered by the STEP trial. Buchbinder said that the trial participants would be followed to find out if the apparent extra vulnerability to HIV conferred by the vaccine was long-lasting, and Mark Robertson outlined a whole range of other factors that would be analysed, including other immune activation markers and CD4 subsets, markers of genetic vulnerability to infection such as HLA genotypes, herpes (HSV-2) status, seminal immune-cell subsets and seminal HIV viral load in seroconverters, and so on.