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J Infect Dis. Author manuscript; available in PMC Feb 2, 2010.
Published in final edited form as:
PMCID: PMC2815505
EMSID: UKMS28543

Infant HIV-1 Infection Severely Impairs the Bacille Calmette-Guerin Vaccine-Induced Immune Response1

Abstract

Background

World-wide, most infants born to HIV-infected mothers receive BCG. Tuberculosis is a major cause of death of HIV-infected infants in sub-Saharan Africa, and should be prevented. However, BCG may itself cause disease (BCGosis) in these infants. Information regarding the immunogenicity of BCG is imperative for the risk/benefit assessment of BCG vaccination in HIV-infected infants; however, no such data exists.

Methods

We compared BCG-induced CD4 and CD8 T cell responses, assessed by flow cytometry, in HIV-infected (n = 20), HIV-exposed but uninfected (n = 25), and HIV-unexposed (n = 23) infants, over their first year of life.

Results

BCG vaccination of the 2 HIV-uninfected groups induced a robust response, characterized by IFN-γ, TNF-α, and/or IL-2-expressing CD4 T cells. In contrast, HIV-infected infants had a markedly lower response, throughout the first year of life. These infants also had significantly reduced numbers of IFN-γ, TNF-α and IL-2 co-expressing polyfunctional CD4 T cells, thought to indicate T cell quality.

Conclusions

HIV-1 infection severely impairs the BCG-specific T cell response during the first year of life. BCG may therefore provide little, if any, vaccine-induced benefit in HIV-infected infants. Considering the significant risk of BCGosis, these data strongly support not giving BCG to HIV-infected infants.

Keywords: Tuberculosis, BCG, HIV, infants, T cells, immune response, polyfuntional, Th1, Th17

Introduction

The Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccine is given at birth to almost all infants born in sub-Saharan African countries [1]. Although BCG affords highly variable protection against pulmonary tuberculosis (TB), the vaccine does protect against severe disseminated forms of TB in infancy by an estimated 73% (67-79%) against meningitis, and 77% (58-87%).against miliary disease [1]. However, there is no clear evidence that BCG protects HIV-infected infants against these forms of the disease [2]. In sub-Saharan Africa an estimated 1500 infants are born with HIV-1 infection every day [3]. TB is common in this population: in South Africa, the incidence of TB in HIV-infected infants and children was 9.2% in 2004, and the disease remains one of the main identifiable causes of death in this population [4]. Rather than protect against severe TB, BCG vaccination of HIV-infected infants may cause disease, called BCGosis [5, 6]. BCGosis, estimated to affect 110-417 per 100,000 vaccinees in South Africa [5], can occur in localized or disseminated forms and cause significant morbidity [7]. To guide a more comprehensive assessment of risks and benefits of BCG vaccination in HIV infected infants, we assessed whether BCG induces the immune response thought to be required to protect infants against TB.

The immune determinants of vaccination-induced protection against TB are not fully understood. However, the T-helper type 1 (Th1) cytokine response, characterized by IFN-γ, TNF-α, and IL-2 production, is widely thought to be essential. We have recently shown, in HIV-uninfected infants, that BCG induces CD4 and CD8 populations that express combinations of IFN-γ, TNF-α and IL-2 [8]. These include “polyfunctional” CD4 T cells, i.e., cells that co-express all 3 cytokines [8], considered good indicators of the quality of the immune response. Although evidence in humans remains inconclusive, animal data suggest that polyfunctional T cell responses correlate with protection against TB in novel TB vaccine studies (personal communication, Anderson P, et al). We therefore measured these T cell markers as primary outcomes in this study, in a comparison between HIV-infected and HIV-unexposed, healthy infants.

A secondary aim was to assess whether immunity induced in HIV-uninfected infants born to HIV-infected mothers was similar to that in HIV-unexposed, healthy infants. There is no data on the efficacy of BCG vaccination in HIV-exposed, uninfected children. However, multiple studies have shown immunological changes [9-11] in and increased mortality and morbidity [12, 13] of HIV-exposed infants.

Materials and Methods

Participants

Infants born to HIV-infected and non-infected women, recruited at clinics or maternity wards in the Worcester region of the Western Cape, South Africa, were enrolled between 2003 and 2006. All infants had documented BCG (strain Staten Serum Institute, SSI) vaccination on the first day of life. Maternal HIV status was determined by HIV antibody ELISA prior to enrollment. Infant HIV infection status was determined by viral amplification at 6 weeks of age. Infants were assigned to one of three groups: infants born to HIV-uninfected mothers (in this manuscript termed “HIV−”), HIV-uninfected infants born to HIV-infected mothers (“Exposed HIV−”), or HIV-infected infants (“HIV+”). Antiretroviral therapy (ART) was not routinely available for treatment of HIV infection during the study period; at that stage, the public health service focused on preventing mother-to-child transmission.

The protocol was approved by the Research Ethics Committee of the University of Cape Town, and by the Institutional Review Board of the Rockefeller University (New York, NY) and the University of Medicine and Dentistry of New Jersey. Good clinical practice and ethical guidelines of the US Department of Health and Human Services and the South African Medical Research Council were adhered to; this included written, informed parental consent.

Blood collection and processing

Blood was collected at 3, 6, 9 and 12 months of age into heparinised tubes. This blood was immediately processed at the field site, using whole blood assays previously optimised for this purpose [8, 14]. Whole blood was incubated with BCG and anti-CD28 and anti-CD49d for 12 hours. Blood incubated with co-stimulatory antibodies alone (UNS), served as negative control. After 7 hours, 50-100μL plasma was removed and cryopreserved for soluble cytokine analysis. Brefeldin A (10μg/mL, Sigma) was added for the last 5 hours of incubation. Following incubation, red blood cells were lysed and white cells fixed with FACS Lysing Solution (BD Biosciences), and then cryopreserved. A small aliquot of heparinized blood was stained for CD3, CD4, CD8 and CD45. A full blood count and HIV viral load (only for HIV-infected infants) were performed at each time point.

Antigens and antibodies

Live BCG was reconstituted from the vaccine vial (SSI) at 1.2 × 106 CFU/mL, as previously described [14]. SEB (Sigma) was used as positive control at 10μg/mL. Anti-CD28 and anti-CD49d (BD Biosciences) were each used at 0.5μg/mL. For flow cytometry the following antibodies were used: anti-CD3 Pacific Blue or PE (UCHT1), anti-CD4 allophycocyanin (SK3), anti-CD45 FITC (HI30), anti-CD8 PerCP-Cy5.5 or PerCP (SK1), anti-IFN-γ AlexaFlour700 (B27), anti-IL-2 FITC (5344.111) and anti-TNF-α PE-Cy7 (MAb11), all from BD Biosciences.

Cell counts, staining and flow cytometric analysis

CD4 and CD8 lymphocyte frequencies were determined by flow cytometry upon staining whole blood for CD4, CD8 and CD45. These values were used with results of CD3 staining and lymphocyte counts to calculate absolute CD4 and CD8 T cell numbers per mL blood.

Intracellular cytokines were acquired on a LSRII flow cytometer (BD Biosciences) and analysed as described [8]. Automated compensation with mouse IgG κ-beads and cell controls was applied. Flow cytometric plots are displayed using bi-exponential scaling. Gating strategies are shown in Suppl. Figure 1.

Soluble cytokine analysis

Plasma was thawed to measure levels of IL-2, TNF-α and IFN-γ by sandwich ELISA following the manufacturer’s instructions (BenderMed for IFN-γ; eBiosciences for TNF-α and IL-2).

Data analysis and modelling

Background cytokine levels measured in plasma, and background frequencies of intracellular cytokine expression, from unstimulated blood, were subtracted from levels and frequencies following BCG stimulation. Absolute numbers of BCG-specific cytokine-producing cells were calculated for each time point. These data were imported into SPICE, version 4.1 (kindly provided by Mario Roederer, Vaccine Research Center, NIAID, NIH) and analysed, before exporting to Prism 4.03 (GraphPad) or STATA 9.0 for statistical analysis.

The distributions of absolute T cell counts were skew due to the presence of some large values. We did not exclude any measurements but reduced the possible impact of these extreme values by log transforming the counts. Log-transformed absolute T cell counts were analysed using mixed effects linear regression models to test for group and time effects and for group-time interactions. These mixed effects regression models treat participants as random effects to cope with repeated measures. This has the effect of imposing a non-zero, equal correlation structure on all measurements within a participant. These models were fitted using maximum likelihood estimation and thus observations at each time point influenced estimates of treatment or other effects at every time point including the missing time points through the specification of the correlation pattern [15]. The numbers of infants from each group at the different time points are listed per analysis in each figure legend and the total number of infants per group and time point in Table 2.

Table 2
Participant numbers

Time was modelled as a continuous (as opposed to categorical) effect and either linear or quadratic time profiles were fitted. In this way time profiles were smoothed out. All group effects estimated from these models are thus adjusted for time but we chose not to report time effects in the tables. Total CD4 (or CD8) cell counts were added to the models to adjust group comparisons for differences in CD4 (and CD8) counts (Table 1 and Suppl. Figure 3).

Table 1
Differences in absolute T cell responses between infant groups estimated from mixed effects maximum likelihood regression models (overall effect)

Estimated group differences of log-transformed values were exponentiated to obtain proportional differences between groups with respect to original untransformed measurements (Table 1).

The distributions of the T cell frequency data were extremely skew and log-transformations did not result in symmetrical distributions. As a result, normal-base linear regression type models could not be used to model the frequency data. These measurements were thus summarized by group and time point using medians and interquartile ranges and compared at each time point using Kruskal-Wallis (overall effect) and Mann-Whitney U tests. Resulting p-values from frequency data should be interpreted conservatively because of the increased chance of false-positives due to multiple testing. Furthermore, comparison at later time points may be biased as a result of infant drop-out.

Results

Infant follow-up and losses

Of 298 infants born to HIV-infected mothers, 20 were identified as HIV+ by a positive viral amplification test. The number of infants in the HIV+ group decreased gradually over the 12 month period (Table 2). Eight infants died during the study, conducted before ART was routinely available for treatment of HIV infection in South Africa, and 4 infants were lost to follow-up. Median log10 plasma viral loads in the HIV+ infant group at the four timepoints were 5.7, 5.6, 4.9 and 4.9 RNA copies per mL, respectively. Three infants received ART, and their data from after initiation of ART were excluded from analysis. Twenty five of the 278 non-infected infants born to HIV-infected mothers were included in the Exposed HIV− group. None of these infants were breastfed. Thirteen of these infants were lost to follow-up over their first year or life. Twenty three infants born to HIV-negative mothers (HIV−) were also enrolled. Of these, 4 were lost to follow-up. The data analysis approach employed specifically assessed whether measured outcomes were reliable regardless of the losses to follow-up (see Data analysis and modeling, under Methods).

BCG-specific Th1 cell numbers were reduced in HIV-infected infants

We measured the BCG-induced immune response in infants by quantifying Th1-cytokine expression in BCG-specific T cells by flow cytometry. The BCG-specific response comprised multifunctional CD4 and CD8 populations expressing IFN-γ, TNF-α and/or IL-2 (Suppl. Fig. 1), as reported before [8]. In all three infant groups the magnitude of cytokine-producing cells peaked at 3 months and then waned over the 9-month follow-up period (Fig. 1). The total BCG-specific CD4 T cell response, comprising all cytokine-expressing cells, was significantly lower in HIV+ infants, compared with the other two groups, throughout the follow-up period (Table 1 and Fig. 1A). This pattern was also seen when the absolute numbers of BCG-specific CD4 T cells expressing IFN-γ, IL-2 or TNF-α were quantified (Table 1, Fig. 1B-D). No differences in total cytokine-expressing or IFN-γ, IL-2 or TNF-α-expressing CD4 T cells were seen in Exposed HIV− and HIV− infants.

Figure 1
CD4 T cell cytokine responses induced by incubation of whole blood with BCG, as measured by flow cytometry. (A-D) Absolute (abs.) cytokine-producing CD4 T cell numbers (no.) in the three infant groups. Background values (unstimulated blood) were subtracted ...

In a complementary analysis, we showed that BCG-specific CD4 T cell loss was independent of CD4 T cell counts. Please refer to Suppl. Results.

Specific polyfunctional CD4 T cell responses were compromised in HIV-infected infants

We quantified BCG-induced polyfunctional CD4 T cells, which simultaneously express IFN-γ, IL-2 and TNF-α, in the three infant groups. Absolute numbers of these cells in HIV+ infants were significantly lower than in the other two groups, throughout the first year of life (Table 1, Fig. 2). By 9 months of age the depletion of polyfunctional cells was striking in the infected group; these cells were barely detectable in most HIV+ infants. Again, no differences were observed between Exposed HIV− and HIV− infants (Table 1, Fig. 2).

Figure 2
“Qualitative” differences of BCG-specific CD4 T cell responses in the three infant groups, based on co-expression of type I cytokines. (A) Absolute polyfunctional (IFN-γ+, IL-2+ and TNF-α+) CD4 T cell numbers at each time ...

Next, we compared the proportions of monofunctional (cells producing 1 cytokine), bi-functional (2 cytokines) and polyfunctional CD4 T cells (3 cytokines) in these infant groups. The BCG-specific CD4 T cell response, which was significantly lower in HIV-infected infants than the other groups (Fig. 1), was also predominantly comprised of mono-functional cells (Fig. 2B). HIV− and Exposed HIV− infants displayed markedly higher proportions of bi- and polyfunctional specific CD4 cells than HIV+ infants (Fig. 2B).

CD8 T cells did not compensate for loss of specific CD4 responses in HIV+ infants

We have previously shown that BCG-vaccination induces CD8 T cells, although at a lower frequencies than CD4 T cells [8]. Specific CD8 T cells mostly express IFN-γ, and to a lesser extent TNF-α, IL-2 and cytotoxic markers such as granzymes and perforin [8, 16]. Since the CD4 T cell compartment is compromised in HIV-infected infants, we hypothesized that this loss would be compensated for by the CD8 T cell response. IFN-γ-producing CD8 T cells were detectable in most infants, in all groups; however, TNF-α and IL-2-producing cells were very infrequent and mostly undetectable (data not shown). Regression modeling of absolute BCG-specific IFN-γ-expressing CD8 T cell numbers displayed lower responses in the HIV+ group, compared with the HIV− group (Fig. 3A). Similar patterns were observed when IFN-γ+ CD8 T cell frequencies were analysed (Fig. 3B).

Figure 3
BCG-specific IFN-γ CD8 T cell responses. Background values were subtracted and observed responses are shown as absolute counts (A) and as frequencies (B). For (A), comparisons (overall effect p values tabulated) were calculated from mixed effects ...

BCG-specific secretion of IFN-γ and IL-2 was reduced in HIV-infected infants

To confirm the flow cytometry results, we quantified BCG-specific secretion of soluble IFN-γ, IL-2 and TNF-α in plasma. As seen for CD4 T cell cytokine production, the levels of IFN-γ and IL-2 at 3 months were significantly lower in HIV+ infants compared with exposed and unexposed HIV− infants (Fig. 4A and B). Lower IFN-γ and IL-2 values persisted at later time points in the HIV+ group, though these differences were not always statistically significant (Fig. 4B). TNF-α levels were typically 10-fold higher than IFN-γ and IL-2 concentrations and were not different between the infant groups, across all timepoints (Fig. 4C).

Figure 4
Levels of soluble cytokines in plasma from whole blood stimulated with BCG for 7 hours. IFN-γ (A), IL-2 (B) and TNF-α (C) were quantified by ELISA and background values (unstimulated blood) subtracted. For A, the numbers of HIV-infected, ...

Discussion

To determine whether BCG vaccination is immunogenic in HIV-infected infants, we assessed induction of CD4 and CD8 T cells producing Th1 cytokines, a response thought to be required for control of M. tuberculosis infection. We showed markedly lower numbers of BCG-specific CD4 T cells in HIV-infected infants, compared with uninfected infants, 3 months after newborn vaccination. Further, these numbers dwindled to virtually undetectable levels later during the first year of life. We also showed that the ability of CD4 T cells to simultaneously express IFN-γ, IL-2, and TNF-α, thought to indicate the “quality” of the immune response, was diminished in HIV-infected infants, compared with non-exposed infants. Further, we found that the relatively low BCG-induced CD8 T cell response was also lower in HIV-infected infants compared with uninfected infants, and could therefore not compensate for the loss of BCG-specific CD4 T cell immunity. Overall, our results demonstrate that the BCG-induced adaptive immune response is severely impaired and, by 9 months, virtually absent in infants with HIV infection.

The severely impaired T cell response in HIV-infected infants suggests that BCG may afford very little, if any, vaccine-induced benefit. We cannot exclude that immune mechanisms other than T cells, such as antibody responses, could be involved in BCG-induced protection. However, overwhelming evidence supports the central importance of the immune function we examined. Considering the significant risk of BCG disease in this vulnerable group, our data support the recommendation by the WHO Global Advisory Committee on Vaccine Safety that BCG should not be administered to infants who are known to be HIV-infected [17]. BCG vaccination of infants not infected with HIV remains critically important to prevent disseminated disease [1]. The WHO Committee recommends that vaccination in infants born to HIV-infected mothers be delayed until infection has been excluded by a viral amplification test, usually at 6 weeks of age [17]. Unfortunately, in most developing country settings, there is no guarantee that infants will return to the clinic for this test, or the test may not be routinely available. In these settings, the recommendation remains to vaccinate all infants at birth [17].

Two questions emerge from the WHO recommendations. First, is BCG immunogenic in HIV-exposed, but uninfected infants, and second, what are the effects of delaying vaccination beyond the immediate newborn period in this population? Regarding the first question, multiple studies have suggested that maternal HIV infection may adversely influence immune responses to various microorganisms, in uninfected infants [10-13]. Increased global T cell activation in HIV exposed infants has also been shown [9-11], and these factors may contribute to increased mortality and morbidity reported in this population [12, 13]. We therefore hypothesized that BCG-induced immunity in HIV-exposed but uninfected infants would be inferior to that in infants who were not exposed to HIV. However, we found no difference in immunity between these two infant groups. Our data imply that the BCG-induced immune response in HIV-exposed but uninfected infants appears normal, and that this group would benefit from vaccination. Our findings are in agreement with another study showing no significant differences in BCG-specific IFN-γ release measured by ELISA in 7-day whole blood assays in exposed, HIV-uninfected infants when compared with unexposed infants, at six weeks of age [11]. However, when these authors used PPD as antigen for the same analysis, a lower IFN-γ response in Exposed HIV− infants was observed.

We are currently addressing the second question posed above by comparing longitudinal immunity induced when HIV-exposed infants are vaccinated at birth and at 10 weeks of age.

Our study is also the first to describe the kinetics of the BCG-induced immune response in infants in some detail. Given our pre-selected time points, the CD4 T cell response peaked at 3 months post-vaccination, and waned over time. However, BCG-induced protection against disseminated forms of TB persists until at least five years of age [1]. This may imply that, after contraction of the peak response measured at 3 months of age, central memory T cells may migrate from the blood to lymph nodes or specific organs, resulting in a quantitatively lower response measurable in peripheral blood. These observations underline the importance of studies of BCG-induced immune correlates of protection.

Interestingly, the proportion of CD4 T cells co-expressing two or three cytokines decreased over time in all groups. Presence of these polyfunctional T cells may indicate “quality” of the immune response, as in HIV infection, presence of HIV-specific polyfunctional T cells is associated with better clinical outcome [18, 19]. Similar observations have been made in murine models of Leishmania infection [18, 19], and of M.tb infection [19] (personal communication, Anderson P, et al). A similar evolution from a high to a low proportion of polyfunctional T cells was reported for Ag85A-specific CD4 T cells after vaccination of healthy adults with a novel TB vaccine, MVA85A [20]. A polyfunctional response may therefore reflect an “effector” response soon after antigen exposure whereas maturation of T cells may be associated with differential cytokine profiles. Alternatively, polyfunctional T cells may have redistributed to lymph nodes or specific organs.

Findings from T cell analysis were confirmed when soluble IFN-γ and IL-2 were measured in plasma, which were significantly decreased in the HIV+ infant group. The comparable levels of soluble TNF-α levels between the different infant groups is likely due to TNF-α production by BCG-reactive innate cells such as monocytes, neutrophils and NK cells. Indeed, soluble TNF-α concentrations exceeded IFN-γ and IL-2 concentrations approximately 10-fold, suggesting an additional source of this cytokine.

The significant drop-out rates in the HIV-infected infant group, primarily due to death, constituted a limitation of our study. These death rates are consistent with those observed in other African infant cohorts not on ART [21]. We therefore employed maximum likelihood mixed effects linear regression modelling, which is designed to cope with missing data points. These models generated similar results to those observed when less sophisticated, non-parametric tests were applied. Another limitation was that no data was available from BCG-unvaccinated infants. Such data would confirm that T cell responses are absent in BCG-naïve infants, and thus that our assay system detects specific T cells. Since BCG is routinely given to all South African infants on the first day of life, analysis of BCG-unvaccinated infants was not possible. However, in another study by our group, utilising an identical assay to the one used here, we detected no BCG-specific T cell responses in BCG-naïve infants from South Africa (Kagina and Hanekom, unpublished).

We did not prospectively address co-variates that could have affected infant immunity, such as maternal viral load and immune status, maternal therapy, infant nutritional status, and additional infections. Regardless, the BCG-specific T cell responses in HIV-infected infants remained strikingly different to the two control infant groups.

In conclusion, our findings suggest that BCG vaccination may not protect HIV-infected infants against TB. How can protection against TB then be achieved in HIV-infected infants? One approach may be to institute highly active antiretroviral therapy as early as possible in these infants, and thereafter vaccinate with BCG. Evidence is emerging that early ART of HIV-infected infants significantly decreases mortality, compared with initiation of treatment when indicated by clinical or CD4 T cell criteria [22-24]. Moreover, the course of BCG-related illnesses including BCG-associated immune reconstitution inflammatory syndrome in HIV-infected infants is less severe when treatment is initiated early [22, 24]. Regardless, the approach of BCG vaccination following early ART would have to be studied formally. An alternative approach is to institute isoniazid prophylaxis in all HIV-infected infants. This intervention has been shown to significantly reduce mortality and the incidence of TB in HIV-infected infants and children [4]. Many questions surrounding this intervention remain unanswered, such as the effects of isoniazid on drug resistance in populations. Early isoniazid prophylaxis is therefore the focus of large trials currently underway. Ultimately, a novel TB vaccine which is safer and hopefully more effective than BCG in HIV-infected infants, will constitute the most sustainable intervention.

Supplementary Material

Acknowledgements

We thank the infants and mothers who took part in this study, their families, and the support of the excellent team at our field site

Funding

This work was primarily supported by the Elizabeth Glaser Pediatric AIDS Foundation and the TBRU of the NIH (NO1-AI70022). TJS is a Wellcome Trust Research Training Fellow (080929/Z/06/Z). WAH is also supported by the NIH (RO1-AI065653), the Aeras Global TB Vaccine Foundation and the European and Developing Countries Trials Partnership (EDCTP). GK was supported in part by the NIH (RO1-AI054361).

Footnotes

1Conflict of Interest Statement

None declared.

This work has, in part, been presented at the 7th International Conference on the Pathogenesis of Mycobacterial Infections, Stockholm, Sweden, 2008 (Abstract O19).

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