Antiretroviral therapy interruptions result in loss of protective humoral immunity to neoantigens in HIV-infected individuals
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AIDS: 17 July 2012
"Importantly, the rate of decline of the protective titers was also significantly greater in the intermittent ART group......
the temporary loss of CD4+ due to acute viremia during ART interruption is unlikely to be directly responsible, but the effect of short-term viremia (and subsequent inflammation and T-cell activation) on the bone marrow microenvironment should be considered.... patients on intermittent ART (arm 2) lost protective titers at a significantly higher rate than control individuals (P = 0.0029), further confirming that individuals undergoing cyclical ART interruptions lose antibody titers to a neoantigen at a faster rate than individuals who maintain suppressive ART over time"
Azzoni, Livioa; Foulkes, Andrea S.b; Firnhaber, Cynthiac; Yin, Xiangfana; Xiang, Zhi Q.a; Li, Yana; Stevens, Wendyd; Gross, Roberte; Ertl, Hildegund C.J.a; Sanne, Ianc; Montaner, Luis J.a
aThe Wistar Institute, Philadelphia, Pennsylvania bDivision of Biostatistics, University of Massachusetts, Amherst, Massachusetts, USA cClinical HIV Research Unit, Faculty of Health Sciences, Department of Medicine, University of the Witwatersrand dDepartment of Molecular Medicine and Haematology, University of the Witwatersrand and the National Health Laboratory Services, Johannesburg, South Africa eDepartments of Medicine (Infectious Diseases) and Biostatistics and Epidemiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Objective: Sustained antiretroviral therapy (ART)-mediated viral suppression restores responses to vaccination in HIV-1-infected individuals. As ART interruption occur frequently in resource-constrained settings, we studied their effects on the ability to mount humoral immune responses against a neoantigen.
Design: Treatment-naive HIV-1-infected individuals were treated with stavudine, lamuvidine and lopinavir/ritonovir. Individuals who maintained viral load less than 50 copies/ml and CD4+ T-cell counts more than 450 cells/μl for 6 months received three doses of rabies vaccine, and were randomized to 72 weeks of continuous ART (arm 1) or sequential 2, 4 and 8-week ART interruptions (arm 2). An additional vaccine dose was administered at study end.
Methods: Neutralizing antibody titers to rabies virus were assessed in plasma with a rapid fluorescent focus-inhibiting test.
Results: The proportion of participants achieving protective (>0.5 IU/ml) antibody titer after vaccination was similar (arm 1 = 92%; arm 2 = 91%), but over time the cumulative proportion of observations with protective titer was greater in arm 1 than arm 2 (P = 0.0177). From week 26 after vaccination, antibody titers were lower in arm 2 than arm 1, and volunteers in arm 2 lost protective antibody titers at a greater rate (P = 0.0029). After boosting, 100% of arm 1 and 95% arm 2 volunteers achieved protective antibody titer.
Conclusion: Our data indicate that individuals undergoing recurring ART interruption retain lower neutralizing antibody titers to a neoantigen, but maintain the ability to mount secondary responses upon boosting, suggesting that they might benefit from vaccine schedule intensification.
Sustained antiretroviral therapy (ART)-mediated viral suppression improves immune responses to vaccinations in HIV-1 infected individuals [1–4]. ART has been available in resource-constrained countries for several years through governmental and international funding programs , and adherence to ART in sub-Saharan Africa has been high . However, supply chain interruptions, stock outs, power outages, employment migration, conflicts, and significant cultural stigma can disrupt adherence in these settings [7–9], with reported rates of therapy interruptions in regular therapy management in sub-Saharan Africa of 12.8 per 100 person years . Qualitative studies have assessed the barriers to access to care [11,12] and demonstrated that instability and conflict result in lower adherence in children , whereas also pointing out that populations in conflict areas can be served effectively .
Although the negative correlates of protracted ART interruption have been characterized in a number of recent studies, ranging from increased rates of opportunistic infections, cardiovascular disease and ART resistance [15–19], it remains to be established how repeated, short-term interruptions, as are likely to occur in clinical settings, impact the levels and quality of overall immune reconstitution obtained while on ART. We recently reported that brief (up to 8 week) interruptions of ART do not result in permanent CD4+ cell loss ; however, in our study, patients undergoing ART interruptions forfeited the gains in CD4+ cell count observed in control individuals on continuous ART.
The relationship between viral replication and the establishment and maintenance of B-cell memory remains unclear. Early reports evidenced that chronic HIV infection causes polyclonal B cell activation, with resulting hypergammaglobulinemia . B cell memory subsets are altered with expression of markers indicative of cell exhaustion, and responses to neoantigens are impaired (reviewed in ) as recently demonstrated in a cohort of viremic HIV-infected individuals with low CD4+ cell count receiving rabies vaccination .
In primate models, Kuhrt et al. demonstrated that naive B cells are lost quickly upon simian immunodeficiency Virus (SIV) infection, and their recovery after ART initiation is delayed as compared to the recovery of IgDneg memory B cells.
Recently, Gelinck et al. demonstrated that patient undergoing ART and with a CD4+ cell count more than 500 cells/μl had incomplete immune reconstitution, but recovered the ability to mount a full antibody response to CD4+-dependent antigens and develop protective immunity upon receiving a course of rabies vaccination. In prior clinical studies  we demonstrated that T-cell-mediated responses to recall antigens were not affected by brief (up to 12 weeks) viremic episodes.
In contrast to B and T lymphocyte subsets, the effects of short-term viremic episodes on the maintenance of antibody titers and long-term B cell memory in ART-treated individuals (i.e. individuals who have recovered the capability of mounting a satisfactory B-cell-mediated response) is less clear.
To assess their impact on immune fitness, we studied the effects of recurring ART interruptions on the ability to maintain protective antibody titers against a neoantigen by comparing the antibody titers to a full rabies vaccination course in individuals who, after receiving the same ART regiment for 6 months and achieving a CD4+ cell counts of 450 cells/μl, were randomized to continuous or intermittent ART.
We have assessed antibody titers to a neoantigen (rabies vaccine) over the course of a cycle of short-term ART interruptions in HIV-infected patients.
Our data indicate that upon undergoing sustained (6 months) viral suppression and achieving a partial immune reconstitution (CD4+ >450 cells/μl), most individuals in our cohort developed a satisfactory B-cell-mediated response to the neoantigen. This observation is in keeping with reports that ART-mediated viral suppression results in increased vaccine responses . Because the vast majority of individuals had similar neutralizing antibody titers, we conclude that adequate B and CD4+ T-cell responses existed in both arms at the time of vaccination.
The titers of neutralizing antibody to rabies virus were lower, at most time points (including after a 2-week interruption), in the intermittent ART arm as compared to controls on continuous ART. Importantly, the rate of decline of the protective titers was also significantly greater in the intermittent ART group.
A detailed analysis of the pathogenetic mechanisms underlying the observed loss of antibody titers is beyond the scope of this work; however, it's important to remark that although CD4+ T cells are necessary to establish a full response to the rabies vaccine , previous work failed to report a correlation between CD4+ cell counts and long-term serum antibody titers to vaccinia  or measles . In addition, our own analysis failed to demonstrate a significant effect of initial CD4+ cell count and viral load, as well as the CD4+ cell count at the time of vaccination on the antibody titers achieved in response to vaccination. Thus, the temporary loss of CD4+ due to acute viremia during ART interruption is unlikely to be directly responsible, but the effect of short-term viremia (and subsequent inflammation and T-cell activation) on the bone marrow microenvironment should be considered. Although memory B cells residing in the marginal zone of spleen and the subepithelial regions of lymph nodes  are considered the initiators of recall responses, protective antibody titers are maintained by long-lived antibody secreting plasma cells that largely home to the bone marrow (reviewed in ). A number of factors derived from bone marrow stromal cells have been implied in promoting the survival of plasma cells (e.g. IL-6, VLA-4 and CD44) . In addition, long-lived plasma cell survival in the bone marrow requires signaling through the tumor necrosis factor (TNF) receptor family member B cell maturation (BCMA) . Prior findings indicating that expression of TNF-receptor family members BCMA, B Lymphocyte Stimulator (BLyS) receptor B cell-activating factor receptor (BAFF-R) and CD95 is altered in viremic HIV-infected individuals  leading to a proapoptotic bone marrow environment.
We postulate that the reported alterations of the levels of BCMA, BLyS receptor (BAFF-R) and CD95 in viremic individuals , which is believed to result in a proapoptotic state, may contribute to deplete long-lived plasma cells from bone marrow even during short-term viral replication, contributing to the observed loss of protective immunoglobulin (Ig) titer, but not B-cell memory.
Polyclonal B cell activation may also contribute to the loss of antibody titers in individuals experiencing brief viremic episodes [21,40]. A loss of memory B cells , which has been associated with loss of specific Ig titers , did not seem to occur in our study, as recall responses to vaccine boost appeared to be similar in both groups. Further clinical studies incorporating bone marrow sampling will be required to determine if BCMA regulation and plasma cell apoptosis are indeed causative for the observed loss of antibody titers.
Administration of a recall vaccine dose at the end of the trial resulted in a protective titers in most individuals in both arms, and no significant difference in mean antibody titer was observed between arms at this point. This may be related with the rapid recovery of B cell memory subsets observed by Kuhrt et al. in primate models of SIV infection and suggests that B cell memory is not irreparably lost upon brief episodes of viremia, and can be rapidly restored upon viral resuppression. Taken together, these data suggest that the adverse effect of brief viremic episodes may be limited to plasma cells and/or circulating antibody titers, the level of which appears to decay faster in individuals experiencing ART interruptions.
If confirmed in larger cohorts, our results have potential clinical implications, suggesting that the loss of vaccine-induced protective antibody titers should be considered when assessing individuals’ adherence, and in the course of treatment interruptions dictated by the management of adverse events and other clinical situations. If a vaccination (e.g. influenza) is administered in individuals that subsequently interrupt ART or have poor adherence, the titers might need to be reconfirmed over time, so that recall vaccination may be scheduled as needed to avoid the loss of protective antibody titers.
Importantly, our study does not address the potential effects on poor adherence and/or ART discontinuations on the effectiveness of childhood immunizations administered according to predetermined priming and boosting schedules; further studies will be required to assess the effect of ART discontinuation in these individuals.
This study has some limitations. In the first place, the study was limited to a single T-cell-dependent immunization, to leverage the low frequency of prior exposure to rabies vaccination (neoantigen) in the target population. Further studies, beyond the scope of this report, should address multiple neo and pre-existing responses (e.g. to seasonal flu vaccine and cytomegalovirus), comparing the respective antibody titers to address time-dependent fluctuations.
Moreover, the number of individuals analyzed is limited. Although we consider this number sufficient for the exploratory nature of this study, we cannot exclude that small differences in antibody titers after primary or recall vaccination might be detected in larger studies.
Finally, our cohort started treatment with a CD4+ cell count of 250–350 cells/μl, and was, therefore, not very immune compromised at the outset. Further studies may benefit from a stratification of individuals with lower and higher CD4+ cell counts as reflective of more or less advanced disease.
Effectiveness of vaccination
As summarized in Table 1, prior to vaccination most participants had low to undetectable levels neutralizing antibody to rabies virus (mean log10 titers were -1.75 ± 0.6 for arm 1 and -1.87 ± 0.36 for arm 2); these values were not significantly different between arms.
After three vaccine inoculations, 21 of 25 participants in arm 1 and 21 of 23 participants in arm 2 achieved protective neutralizing antibody titers (≥0.5 IU/ml), as reflected by a mean log10 titer of 0.18 ± 0.57 for arm 1 and 0.09 ± 0.36 for arm 2.
As described in our prior report of the study endpoints , CD4+ cell counts, which were similar at baseline and at the time of vaccination, were significantly lower in arm 2 than arm 1 during or immediately after an ART interruption (study week 32 and 76, P = 0.0202 and 0.0003 respectively, Table 1), but were not significantly different at other time points (e.g. study weeks 48, 64 and study end).
We assessed the effect of baseline CD4+ cell count, baseline viral load and vaccination time CD4+ cell count on log10 neutralizing antibody titer using a linear model; none of these variables had a significant effect.
Effect of antiretroviral therapy interruptions on antibody titers
To assess the effect of cyclic treatment interruptions on the retention of neutralizing antibody titers to rabies virus, we assessed the overall percentage of observations with protective titer over the observation time until the vaccine boost (study weeks 32, 48, 64/68 and 80/76). As illustrated in Fig. 2a, the overall proportion of participants with protective titers was greater in control individuals than in patients with intermittent ART (arm 1, mean 0.47% ± 0.36; arm 2, mean 0.22% ± 0.35, P = 0.0177).
As antibody titers naturally decrease over time, we assessed whether the proportion of individuals with protective titers were different between arms at different postvaccination times. As summarized in Table 1, the proportion of individuals with protective titer was lower in the intermittent ART group, and this difference reached statistical significance at study week 80, when 74% of individuals in arm 1 had protective titers, as compared to 24% in arm 2 (Fisher exact test P = 0.0022).
Accordingly, a direct assessment of the antibody titers indicated that these were significantly higher control individuals at multiple postvaccination time points (mean log10 titer at week 48: arm 1 -0.58 IU/ml ± 0.70, arm 2 -1.13 ± 0.88, P = 0.0236. Weeks 64/68: arm 1, -0.74 ± 0.76, arm 2, -1.21 ± 0.83, P = 0.0459. Weeks 80/76, arm 1 -0.25 ± 0.66, arm 2 -1.2 ± 0.92, P = 0.0004), further supporting a loss of antibody titer over time.
Finally, we assessed the proportion of participants in each arm who, once they lost protective antibody titer, failed to recover it until booster immunization using a log-rank test. As illustrated in Fig. 2b, patients on intermittent ART (arm 2) lost protective titers at a significantly higher rate than control individuals (P = 0.0029), further confirming that individuals undergoing cyclical ART interruptions lose antibody titers to a neoantigen at a faster rate than individuals who maintain suppressive ART over time.
We also assessed the effect of baseline CD4+ cell count, baseline viral load and vaccination time CD4+ cell count on the time to failure using a linear model; none of these variables had a significant effect, suggesting that baseline conditions do not affect the loss of antibody titer during interruption.
We sought to determine whether cyclical ART interruptions had lasting effects on antibody recall responses. To this purpose, qualifying participants (ART resumed, viral load <400) were inoculated with one dose of rabies vaccine at study end. Samples for this assessment were available for 20 participants in arm 1 and 22 in arm 2; individuals missing this time point had similar baseline titers as those assessed.
As indicated in Table 1, after receiving the boost, similar proportions of study participants in both study arms (arm 1: 25/25 participants; arm 2: 19/20 participants, P = 0.4444, Fig. 2b, right box) achieved protective neutralizing antibody titer, indicating that the ability to mount a recall response to the antigen was not lost in individuals undergoing cyclical ART interruptions.