Overall, 2 of 48 NVS (4.2%), 8 of 34 LVL (22.2%), 0 of 18 MHVL (0%), and 0 of 18 HAART (0%) individuals had broadly neutralizing antibodies. These 10 carried a diagnosis of HIV-1 for a median of 13 years (range 1-22). Their median CD4 count was 589 cells per microliter (range 202-927) and HIV-1 viral load of 1611 copies per milliliter (range 110-8964).
Six of these 10 individuals with broadly neutralizing plasma continue to maintain normal CD4 counts without therapy. However, HAART was initiated in 4 individuals (3 in the LVL group and 1 in the NVS group). One patient, 14 years after diagnosis, had an increase in HIV-1 RNA from a baseline of 5000-10,000 copies per milliliter to close to 30,000 copies per milliliter with a CD4 loss from 507 to 288 cells per microliter in 6 months. Two patients, although maintaining HIV-1 RNA <5000 copies per milliliter, had slow but progressive CD4 loss to less than 300 cells per microliter requiring HAART therapy. The final patient, although maintaining HIV-1 RNA <200 copies per milliliter over many years, likewise had a slow CD4 loss over and HAART therapy was initiated at a CD4 count of 489 (CD4% of 18) 19 years after diagnosis of HIV-1.
Correlation of the Presence of Broadly Neutralizing Antibodies With Viral Load
The presence of broadly neutralizing antibodies was analyzed according to cohort and HIV-1 viral load. Although all of the patients with broadly neutralizing antibodies were in the NVS and LVL groups, there was no correlation between presence of broadly neutralizing plasma and patient cohort (data not shown). However, there was a significant correlation between plasma viral load and the presence of broadly neutralizing antibodies in those with HIV-1 RNA between 102 and 104 copies per milliliter compared with those <102 and those with >104 HIV-1 RNA copies per milliliter (P = 0.0003 and 0.0245, respectively) with the 2-tailed Fisher exact test. In addition, individuals with HIV-1 RNA between 102 and 104 copies per milliliter had a higher number of Tier-2 viruses neutralized compared with the <102 or >104 copies per milliliter groups. This reached significance when compared with the <102 group (P ≤ 0.0001), and there was a trend toward significance in the >104 group (P = 0.076) with the 2-tailed Mann-Whitney test. The correlation of broadly neutralizing antibodies with viral load, and the correlation between the total number of Tier-2 viruses neutralized for the 102-104 compared with the <102 group was significant whether or not the HAART-treated patients were included in the analyses (P = 0.0018 and P = 0.0002, respectively) (Fig. 1).
In addition, individuals with HIV-1 RNA between 102 and 104 copies per milliliter had a higher mean ID80 neutralization titer for each of the 15 viruses tested compared with the <102 or >104copies per milliliter groups. This reached significance for 46.7% of the individual viruses tested when analyzed by single patient neutralization using the 2-tailed Mann-Whitney test. The above is seen in Table 4.
Using logistic regression, there was no relation between presence of broadly neutralizing antibody and age, HIV risk factor, duration of HIV infection, HCV status, or CD4 count (data not shown). There was, however, an association with male sex (P = 0.016), as 10 of 11 persons with broadly neutralizing plasma were male.
The data suggest that in HIV-1-infected individuals, the ability to produce broadly neutralizing antibodies correlates with plasma viremia. The LVL group, which was defined as having viral loads between 500 and 20,000 copies per milliliter, had the most individuals with HIV-1 broadly neutralizing plasma. In addition, there were 2 individuals in the NVS cohort who had broadly neutralizing activity. Importantly, both of these NVS individuals had low-level viremia detectable by routine HIV-1 RNA testing, as opposed to the majority of NVS who consistently have undetectable viremia and do not have broadly neutralizing antibodies. Because of this observation, the data were analyzed according to HIV-1 RNA copies, and the presence of broadly neutralizing antibodies correlated with a viral load of 102 and 104 copies per milliliter. We did not observe any persons with broadly neutralizing antibodies who had <102 or >104 HIV-1 RNA copies per milliliter. This suggests that an optimum amount of antigen production is needed for this response, which is less likely to be seen in patients with higher or lower HIV-1 RNA copies.
Patients with <102 HIV-1 RNA copies per milliliter, whether due to natural control or HAART, demonstrated diminished ID80 neutralization values, an observation made in prior studies.2,7 Likewise, there were no individuals with broadly neutralizing activity in this group. Overall, the low neutralization titers in the NVS and those on HAART suggests that a decrease in antigen load led to a decrease in antibody titer, a situation that has been described in patients after starting HAART.2 The lack of continual high titers in these situations may mirror the poor long-term immunogenicity of gp120 seen in the VaxGen vaccine trial.1 Importantly, a low-plasma neutralization titer in an NVS patient does not necessarily mean that they never had a robust humoral response to HIV. As we have shown in the NVS, a patient can harbor a large number of memory B cells that can be stimulated to produce epitope-specific HIV antibodies that cannot be found in circulation.8
Several previous studies in LTNPs noted increasing neutralization breadth and titers to heterologous viruses over time.9-11 Importantly, not all studies have shown this.12,13 LTNPs, which were defined by CD4 count only, have wide ranging viral loads, and the combined LVL and NVS groups in this study can be viewed as constituting a cohort similar to LTNPs (though not all would meet the CD4 or time requirements). More recent studies have focused on cohorts of patients, such as ours, that have a definition of HIV suppression based on viral load and not CD4 count.2,7,14,15 In this study, 12% of the combined NVS/LVL groups had broadly neutralizing plasma, which is comparable to the 12% reported for a combined cohort of elite and viremic controllers (similar to our definition of NVS and LVL).14 The disparate results in this study between those NVS who have greater or less than 102 HIV-1 RNA copies per milliliter may account for the discrepancies in prior LTNP studies.
At the high levels of plasma viremia, a diminished humoral response was observed. The ID80 titers in those with >104 HIV-1 RNA copies per milliliter for the Tier 1 and Tier 2 viruses were lower than those with 102-104 HIV-1 RNA copies per milliliter but comparable with those with those with <102 HIV-1 RNA copies per milliliter. Also, there were no persons in this group who demonstrated broadly neutralizing plasma. These data are consistent with the observation of low antibody titers and neutralization breadth seen in the later stages of HIV,10,11,17 which could be due to progressive B cell dysfunction caused by HIV-1.
Recently, some groups have noted increasing neutralization breadth with higher viral loads.16,18-20 These studies have had fewer patients than this study, and most of them have tested fewer viruses than ours. However, it is likely that some of these patients do develop broadly neutralizing antibodies, which are lost as HIV disease progresses. It should be noted that 1 of the 10 patients with broadly neutralizing antibodies in this study (who also eventually received HAART) experienced an increase in HIV-1 viral load to close to 30,000 copies per milliliter. In this individual, we retested the plasma from the time point just before starting HAART and broadly neutralizing antibody response with a similar neutralization pattern to what was seen from the time point 18 months prior. Thus, the development of broadly neutralizing antibodies in this individual was related to the 13 years his immune system was exposed to HIV at lower viral loads (rather than the six months with higher viral loads), and that without HAART, these antibodies could have disappeared with further HIV-mediated immune dysfunction and destruction.
Besides the role of the optimum amount of antigen associated with broadly neutralizing plasma highlighted in this study, duration of infection has also been noted to be important.9,10,16 In this study, the patients with broadly neutralizing antibodies also had been infected with HIV for a relatively long time (a median of 11 years since HIV diagnosis), but we were unable to show an association with time since diagnosis. Furthermore, this cannot be considered the sole determinant, as the NVS and LVL had comparable length of HIV infection, but a difference in HIV-1 RNA copies and ability to neutralize various strains of HIV-1. We were unable to find other correlations with broadly neutralizing plasma other than the viral load and male sex. Regarding the later, 90% (9 of 10) of the patients with broadly neutralizing plasma were male; however, the significance of this association remains unknown.
The study's strengths include the total number of patients tested (120 patients), the high number of Tier 1 and Tier 2 viruses tested (at least 15 viruses per patient). In addition, although we used a definition of broadly neutralizing that has been used previously,6 these were verified with both IgG and heterologous clade testing. Finally, this study was focused on individuals with LVLs (82 NVS/LVL), where the majority of patients with broad neutralization were found. However, our findings need to be considered within the inherent limitations of a cross-sectional study. In terms of generalizability, it is important to note that the study subjects are predominantly African American (>95%), Clade B infected, and have relatively high rates of IDU, thus the findings need to be confirmed in other cohorts of patients. In addition, although we have demonstrated a correlation between viral load and broadly neutralizing antibodies, this study was not designed to find causality or a putative mechanism (such as low antigen stimulation leading to low neutralization levels or high viral loads causing B-cell dysfunction). Finally, correlations about the potential protective effects of neutralizing antibodies cannot be done in this type of study. More prospective studies starting in acute HIV infection, such as the one by Piantadosi et al,18 should be able to answer this question.
In conclusion, these results indicate that low but persistent HIV antigen expression correlates with high levels of broad HIV-1 neutralizing antibody activity. The presence of broadly neutralizing activity was associated with 102 and 104 copies of HIV-1 RNA. At higher levels of plasma viremia, neutralization levels were diminished. Conversely, at lower levels, either due to antiretrovirals or natural suppression by other mechanisms, there seems to be insufficient antigen stimulation to maintain high neutralization levels. These findings may have important implications in furthering the understanding of the humoral response to HIV infection.
We would like to thank members of the NVS Study and Becky Boyce, RN, the study coordinator.
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Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
broadly neutralizing antibody; elite controller; HIV; HIV RNA; natural viral suppressor; neutralizing activity