CROI: Are neutralising antibodies the key to superinfection?

A weaker than usual neutralising antibody response may be the reason why superinfection can occur in recently infected individuals, according to two studies presented earlier this month at the Thirteenth Conference on Retroviruses and Opportunistic Infections (CROI) in Denver. Another two studies presented at CROI provide more insight into superinfection and all four studies have implications for both HIV-positive individuals and preventative vaccine research.

Systemic vs. localised superinfection

In the past, superinfection was described as the reinfection of an HIV-positive person with a genetically divergent strain of HIV to that seen during initial infection.

However, it is now thought that one (or possibly more) subsequent HIV strain(s) can re-infect an HIV-positive person but remain compartmentalised, or localised, most likely within the cells of the genital tract and/or rectal mucosa.

When a subsequent HIV strain becomes the dominant strain of HIV in the body, this is known as systemic superinfection. However, when a subsequent HIV infecting strain remains compartmentalised, it is known as localised superinfection.

Neutralising antibodies

Neutralising antibodies are produced by the immune system's B-cells. They are literally able to neutralise HIV's ability to attach to CD4 T-cells. Research into these neutralising antibodies is still in its infancy; currently between 15-20 such antibodies, which are directed against at least six different regions of HIV, have been discovered.

Much of the research into neutralising antibodies has been undertaken by scientists working on vaccines that are hoped to protect uninfected individuals against HIV at some point in the future.

There had been concern that neutralising antibodies were very specific, and that they may only neutralise a person's own (autologous) HIV strain. The studies below suggest that neutralising antibodies can be cross-protective, providing important information for vaccine development.

However the data on superinfection presented at CROI this year are also of great interest to HIV-positive individuals who are concerned that superinfection may be a clinically significant event, as has been suggested by individual case reports.

The UCLA/UCSD data

Two years ago at CROI, investigators from the University of California, in Los Angeles (UCLA) and San Diego (UCSD) reported three cases of superinfection in a cohort of 78 recently infected individuals that occurred between five to 13 months after the estimated date of initial infection.

The investigators subsequently theorised that it may be a lack of neutralising antibodies in these three men that made them vulnerable to superinfection, and presented the results of their study this year during an oral session in Denver.

They compared the neutralising antibody response before and after superinfection in the three superinfected men they'd previously identified with the neutralising antibody response in a control group of eleven recently infected gay men with continued exposure to HIV and no superinfection. No-one was on antiretroviral therapy.

Neutralising antibody responses were measured against each individual's own HIV strain(s) as well as two lab-made HIV strains (NL4-3 and JRCSF).

They found that prior to becoming superinfected, the superinfected men lacked detectable neutralising antibodies to the two lab-made HIV strains, which was significant when compared with the non-superinfected group, which had stronger responses to the two strains (NL4-3, p = 0.02; JRCSF, p = 0.05). In addition, just one of the three superinfected men showed a (weak) response to his own HIV, unlike the control group, which showed stronger antibody responses to their own HIV (p = 0.09).

After superinfection, neutralising antibody responses to the same three viruses were stronger in the superinfected men, but still not as robust as in the control group.

The investigators also tested the superinfected men to make sure that their lack of neutralising antibody response wasn't because they'd had less time to produce neutralising antibodies after being infected (and superinfected) compared with the control group. In fact, they found the opposite: a trend for the superinfected men to have had their infection longer than the control group, which further underscored the superinfected men's lack of neutralising antibodies.

Dr Davey Smith, presenting, told the CROI audience that the superinfected men had less cross-protective and autologous neutralising antibody responses than the non-superinfected controls. "These data identify cross-protective neutralising antibody in the prevention of superinfection," he concluded, "and is perhaps very important for a protective vaccine design".

The San Francisco data

A second presentation from San Francisco's Gladstone Institute and the University of California, San Francisco (UCSF), appeared to confirm these findings. They had previously identified four cases of apparent superinfection in a cohort of 104 recently infected individuals. Three of the cases occurred within a year of their initial HIV infection; the fourth occurred some time between two and a half and three and a half years later.

They termed these case 'apparent', because they weren't able to definitively rule out that these men were infected with two divergent HIV strains at the same time (known as dual infection). In their presentation of the data at CROI last year (Grant 2005), they reported that virological analysis did not detect the subsequent virus at baseline, but acknowledged that their methods could not detect minority HIV populations of less than around 3%. Without identifying the HIV in the source partners (which was impossible due to the fact that they were anonymous sexual partners unreachable via contact tracing), they had to concede these cases were 'apparent' superinfection.

When the San Francisco researchers compared the neutralising antibody response in the four apparently superinfected individuals with the neutralising antibody response of eight couples with acute or recent HIV infection without evidence of superinfection who were constantly exposing each other to HIV (a median of 200 events of unprotected receptive intercourse) their results were similar to the Los Angeles/San Diego data: they found significantly higher levels of neutralising antibody responses in the non-superinfected couples.

In fact, the non-superinfected couples showed high levels of neutralising antibodies to their own HIV, their sexual partner's HIV, and the viruses of ten other individuals. Dr Jeff McConnell, presenting, pointed out that in most cases neutralisation was stronger to the partner's virus than to their own HIV. This suggests that, in the majority of cases, neutralising antibodies are cross-protective.

Dr McConnell concluded that that since neutralising antibodies "are narrow and weak in those with superinfection" and "are stronger and broader in those exposed but not superinfected", a strong neutralising antibody response may be the reason systemic superinfection does not occur in most individuals.

Implications for HIV-positive individuals

During the question and answer session that followed, Dr Smith spelled out the implications for HIV-positive people. He said that their data suggest that some people are protected against superinfection more than others, and "that there might be a window of opportunity for superinfection to occur, and maybe that is timed by neutralising antibody response. Some people might have neutralising antibody response that is slower and therefore the window is larger."

The Spanish data

A fourth presentation from two Spanish cohorts suggests that superinfection may well be occurring quite frequently in recently infected individuals and that this is reflected by a tiny, but significant, increase in viral load.

This poster presentation included data from two cohorts of recently infected women, and was restricted to the 120 women who filled-in at least one sexual behaviour questionnaire and were naive to antiretroviral therapy (or had only taken monotherapy).

They defined an episode of sexual risk behaviour as "non systematic use of condoms with a partner of HIV-infected or of unknown serostatus," and they considered these women at risk for superinfection. They then compared these 59 women with the 61 women who reported consistent condom use with HIV-positive or unknown partners, or sex with only HIV-negative partners, or no sex.

A total of 51 of the 120 women had a baseline viral load that was below 4 copies/ml, and the investigators focused their attention on these women. They assessed factors that led to a viral load rise above 4 copies/ml and found that the women with a baseline viral load below 4 copies/ml had a mean increase of 0.01 log (about 1 copy/ml) per month (p=0.02) after they first reported unprotected sex with an HIV-positive or unknown status partner. However, the women with a baseline viral load above 4 copies/ml did not have this small but significant increase in viral load after reporting the same risks.

In multivariate analysis the investigators found that the women with a viral load below 4 copies/ml and who were "at risk of superinfection" were about 2.5 more likely (HR 2.31; 95% CI, 0.96-5.52; p=0.06) to experience a viral load increase above 4 copies/ml.

They concluded that this increase in viral load "may be related to superinfection" and that they are currently performing further analysis to identify if this is indeed superinfection.

Do neutralising antibodies keep superinfection localised?

Dr McConnell revealed during the subsequent question and answer session at CROI that his team's preliminary ongoing unpublished research also suggests that neutralising antibodies are preventing systemic superinfection by containing HIV infection locally within rectal mucosa and genital secretions.

A poster presentation by the same group reveals what they know so far, although the poster does not conclude that it is neutralising antibodies that are preventing systemic superinfection.

The investigators examined the DNA of HIV that was archived in the peripheral blood mononuclear cells (PBMCs) of 17 individuals who had been infected with HIV for a median of ten years, as well as two recently infected individuals. All of the individuals were on antiretroviral therapy that suppressed plasma HIV to 'undetectable' levels, and all were being continuously exposed to genetically divergent HIV via sex with their HIV-positive partners.

They found evidence of highly divergent HIV strains in the PBMC DNA of both (100%) of the recently infected individuals, and in three (18%) of the 17 chronically-infected individuals. These HIV strains appeared far more diverse than could be caused by repeated mutation under pressure from the host's immune system alone, suggesting localised superinfection, rather than simply archived mutated strains.

However, HIV strains in three of the five individuals were found to be drug-resistant mutants. Nevertheless, this hadn't caused virological rebound or treatment failure.

They concluded that an unknown mechanism prevents systemic superinfection in chronically infected individuals on treatment. It appears to do this by limiting the HIV strain or strains acquired after the first infection to a localised area. These minority populations are then archived in PBMCs, but even when they are antiretroviral resistant strains they do not appear to cause virological failure. Although the poster did not conclude this, it is possible that this mechanism may be neutralising antibodies.