In previous studies performed among patients seen at the HIV clinic, the Institute of Tropical Medicine, Antwerp, Belgium, a disproportionate number of African HIV-1-infected women were shown to have circulating antibodies that could neutralize different subtypes of primary HIV-1 isolates. However, some relevant factors were confounded because few Europeans were included in the first study , and most of the women in the second study were Africans . The question is whether the disproportionate number of Africans occurs by chance or is a function of their sex or origin.
In this study, 168 plasma samples of African and European men and women (42 of each group) were screened in a randomized manner against primary CA4 (env subtype F), CA13 (env subtype H) from group M and VI686 from group O . Sixteen plasma samples that cross-neutralized all three viruses were further tested against nine other primary isolates, belonging to group M (env A–H) and two of group O.
Neutralization indices (NI) were calculated for each plasma/isolate combination, i.e. the infectious virus titre, expressed in log10, in phytohaemagglutinin-stimulated peripheral blood mononuclear cell cultures divided by the titre after incubation in the presence of plasma. An NI greater than 1.0 (90% reduction) is taken as an indicator for neutralization.
The proportion of NI greater than 1.0 for each isolate tested was used in a binomial expansion to calculate the expected numbers of plasma samples that would neutralize none, one, two and three isolates. The expected values were compared with their observed values for each neutralization pattern and were then compared by χ2 analysis.
Sixteen out of 168 plasma samples (9.5%) neutralized the three isolates. Of these, 15 were Africans (P = 0.006) and one was European. Of the Africans, 10 were women (P = 0.009); the European was a woman.
From the 16 plasma samples that were able to neutralize the three isolates, only six of the African women, one African man and the European woman were able to neutralize 10 or 11 out of 11 of the wider range of isolates (P = 0.06; not statistically significant) (data not shown).
Although plasma from Africans showed a disproportionate number that could neutralize the three isolates, this phenomenom was also observed with the neutralization of one or two isolates (Table 1). The frequencies of neutralizing none, one, two or three isolates by plasma from Europeans showed a good fit with expected frequencies, indicating that the neutralization of one isolate did not influence the neutralization of the others. In contrast, for Africans there was a significant difference between expected and observed frequencies, and this is particularly evident in women.
Cross-neutralization is particularly apparent in the plasma of African women. When plasma were screened against three HIV-1 isolates, no African women had antibodies that were specific for the group O virus nor doubly specific group O and subtype H, although given the numbers of plasma samples that could neutralize these two isolates, seven or eight of the 42 plasma samples should have fallen into one of these two categories.
In contrast, 10 African women were able to neutralize all three isolates against an expected number of two or three. Although there was a similar trend among the African men, the distortion was not so evident because there were six plasma samples that were specific for either the subtype H or group O isolates, such that any difference from an assumed random distribution did not reach statistical significance (Table 1).
These observations are compatible with the idea that neutralization of the subtype H and group O isolates are not independent parameters in African women: the same set of antibodies may be able to neutralize both viruses. This resolves some of the anomalies obtained with previous results.
The disproportionate number of women with broad cross-neutralizing antibodies was probably a result of their being African, rather than because of their sex. In a previous study, we concluded that the neutralizing activity was antibody mediated . Interestingly, Burrer et al.  also observed antibody-mediated neutralization, particularly in African women.
Our results show that there is a difference in neutralization patterns between African and European plasma, especially in African women. An attempt to generate human monoclonal antibodies from African women with broad cross-neutralizing capacity is ongoing.
Guido van der Groen
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