There were some variations in the response profiles for the 7vPnC serotypes between HAART-treated and HAART-naive (Fig. 2a). Although not statistically significant, compared to HAART-naive, HAART-treated patients tended to have a higher response to serotypes 9V, 14, and 18C, but a lower response to serotype 6B. When pooling the seven serotypes, the mean loss of total vaccine-specific IgG from the peak response at month 4 to follow-up at month 9 was significantly greater among HAART-naive than among HAART-treated (54.8 vs. 38.1%, P = 0.001) patients. Boosting with PPV23 did not increase 7vPnC IgG concentrations to levels above those observed 1 month after the second 7vPnC in either group.
Both HAART-naive and HAART-treated had substantial serotype-specific OPA increases following immunization (Fig. 2b). However, 1 month and 6 months after second 7vPnC vaccination, geometric mean OPA titers were higher among those on HAART compared with HAART-naive (statistically significant for serotypes 14, 19F, and 23F). These differences remained significant even with adjustment for baseline CD4+ cell count (data not shown). Boosting with PPV23 increased OPA titers for serotypes 6B, 19F, and 23F to levels above those observed following the second 7vPnC. However, the response curves for serotype 14 showed initial decreases following PPV23 in both groups, which were not observed for the other serotypes.
We found no significant difference in the effect of CPG 7909 on mean 7vPnC IgG-fold increase (from baseline to month 9) between the HAART-treated and HAART-naive patients. We found no significant effect of baseline CD4+ cell count on mean 7vPnC IgG-fold increase from baseline to month 9 in either of the two groups (data not shown). However, among HAART-naive patients who received a vaccine with CPG 7909 as adjuvant, mean 7vPnC IgG increased 2.2-fold (95% CI 1.5–3.3) per log10 HIV RNA decrease (Fig. 3). This effect of HIV RNA was specific for untreated patients receiving CPG 7909 and was not observed among HAART-naive who received a placebo adjuvant.
In this study, we found that HAART and HAART-naive HIV-infected adults with moderate to high CD4+ cell counts had similar vaccine responses initially, but persons on HAART achieved a more durable antibody response of higher magnitude and functional activity following pneumococcal conjugate vaccination than HAART-naive persons, even after adjustment for CD4+ cell count. Thus, HAART-naive patients had greater quantitative loss of vaccine-specific antibodies during follow-up than HAART-treated patients. Further, we found that TLR9-stimulated antibody production correlated negatively with plasma HIV RNA, which indicates that TLR9 adjuvants may not be useful for preventive or therapeutic vaccines in viremic HIV patients.
One of the strengths of this study was the inclusion of both quantitative and functional antibody response as endpoints. Although related, these two parameters reflect different aspects of the immunization response. The ELISA measures total binding IgG antibodies to the capsular antigen, whereas the OPA measures functional activity of the IgG antibody, but clinical data on their correlation with protection against pneumococcal disease in adults are missing. The World Health Organization considers both measurements to be equally important in the evaluation and introduction of new pneumococcal vaccines . This was the first study to evaluate and compare the effect of TLR9 agonist adjuvants in HAART-treated and HAART-naive patients, which allowed us to study the effect of TLR9 stimulation in vivo in viremic HIV patients.
When should persons with HIV be immunized? We found an almost three-fold higher proportion of persistent 7vPnC vaccine responders among HAART-treated compared to HAART-naive individuals. This finding is in line with other recent reports [29,30]. HAART use concurrent with hepatitis B vaccination is associated with a two-fold increased probability of responding to the vaccine . Further, we observed a faster decline in IgG concentrations following pneumococcal conjugate vaccination among HAART-naive compared to HAART-treated patients. This has also been reported following PPV23 in HAART-naive patients  and may be because of the increased turnover of memory B cell in untreated compared with treated HIV patients [15,16]. Only one randomized trial of PPV23 vaccination with clinical endpoints among persons with HIV has ever been conducted. The trial that was conducted among HAART-naive adults in Uganda revealed that there was no increased protection against pneumococcal disease among vaccinees . Interestingly, as demonstrated by adjusted logistic regression and sensitivity analyses, the effect of HAART on vaccine responses cannot be fully explained by differences in CD4+ cell counts. Thus, the increased loss of protective antibodies over time among HAART-naive compared to HAART-treated patients indicates that re-immunization should be conducted after commencement of HAART to achieve optimal and durable protection.
In our unadjusted regression analysis, being current smoker compared with current nonsmoker was associated with a decreased chance of having a persistent vaccine response at 9 months. In the adjusted analysis, the estimate remained similar, but was no longer statistically significant. Others have also observed a negative impact of smoking on pneumococcal vaccination responses . Thus, promoting smoking cessation among HIV patients not only may have a significant impact on their risk of pneumonia  but may also improve the effect of pneumococcal vaccination.
The benefit of combined schedules of pneumococcal conjugate and pneumococcal polysaccharide vaccines is debated . In our study, post-PPV23 IgG concentrations were similar or lower for all 7vPnC serotypes compared with IgG concentrations after second 7vPnC. Others have made comparable observations in different settings , but it remains unknown why we are unable to demonstrate a quantitative booster-effect after 7vPnC in adults, as has been observed in infants. However, looking at OPA titers, it appears that at least for serotypes 6B, 19F, and 23F, the PPV23 booster does seem to have a positive effect, whereas titers for serotype 14 seemed to decrease. A similar OPA response pattern for serotype 14 was observed in a study among children and young adults with sickle cell disease , indicating that vaccine-induced OPA increases for serotype 14 may have a different response profile than the other 7vPnC serotypes. Until recently, OPA was not used in clinical trials because it was technically difficult to perform. Therefore, the clinical significance of OPA titers is not well established, but there are indications that OPA may be better in predicting cross-protection than ELISA measurements. Clinical experience shows that 7vPnC does not induce cross-protection against serotype 19A  and correspondingly OPA titers remain low, whereas levels of antibody measured by ELISA increase . Thus, OPA may be a more accurate reflection of clinical vaccine effectiveness and boosting with 7vPnC or PPV23 may be of considerable value.
The clinical effect of pneumococcal conjugate vaccination in HIV-infected adults is unknown, but hyporesponsiveness – which complicates repeated immunizations with polysaccharide vaccines – does not appear to be an issue with conjugated vaccines . If pneumococcal conjugate vaccines were shown to be clinically effective against pneumococcal disease in adults, they might be more suitable for people with HIV.
We also found that TLR9-stimulated antibody production correlated negatively with plasma HIV RNA. HIV-induced TLR9 stimulation has recently been suggested as a potential driver of chronic immune activation and disease progression . If so, downregulation of TLR9 expression and/or signaling would be a way for the immune system to counteract this pathogenic activation of TLR9. Nowroozalizadeh et al.  demonstrated by whole-blood stimulation that TLR9 responsiveness is decreased in HAART-naive HIV-1-infected compared to HIV-negative individuals and that decreasing responsiveness was positively correlated to increasing viral load. An association between viral load and responsiveness to TLR9 agonist-adjuvanted hepatitis B vaccination was also seen in SIV-infected rhesus macaques – where monkeys with viral loads more than 107 copies/ml were unable to mount an antibody response upon immunization . Thus, individuals with moderate to high level of HIV RNA may not be very susceptible to TLR9 stimulation. This has at least two important implications: TLR9 adjuvants may not be useful for preventive or therapeutic vaccines in viremic HIV patients and these individuals may be more likely to acquire severe infections (like invasive pneumococcal disease ) in which TLR9 signaling is needed in the early stages of the host defense to control infection.
In conclusion, concomitant use of HAART improves and prolongs the antibody response to pneumococcal conjugate vaccines in persons with HIV infection, independently of CD4+ cell count at the time of immunization. Immunization or re-immunization should be conducted after commencement of HAART to achieve optimal and durable protection. Further, the interference of HIV viremia with TLR9-stimulated antibody production may have important implications for the choice of adjuvant in preventive and therapeutic vaccines for HAART-naive persons. More studies are needed to determine the mechanisms responsible for the complex interaction between chronic infection and innate and adaptive immune responses.
The authors thank the participants for their involvement in the trial. They also thank the study nurses, Iben Loftheim and Inge Arbs, for their excellent work as trial site coordinators for the study; Coley Pharmaceutical Group (now part of Pfizer) for providing CPG 7909 for the study; Statens Serum Institut, Copenhagen and Flow Applications Inc., Illinois, USA for conducting the antibody analyses. Grant support by Aarhus University, the Augustinus Foundation, Scandinavian Society for Antimicrobial Chemotherapy, Danielsen's Foundation, AP Moeller's Foundation, Krista and Viggo Pedersen's Foundation, LF Foght's Foundation, KA Rohde, and Hustru's Foundation. Coley Pharmaceutical Group (now part of Pfizer) provided CPG 7909 for the study.
The study was presented in part at the 17th Conference on Retroviruses and Opportunistic Infections in San Francisco, 16–19 February 2010 (abstract 813).
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