Immune responses in patients with HIV infection after vaccination with recombinant Hepatitis B virus vaccine

All the patients and controls received three doses of recombinant Senvac™ HBV vaccine (Shanta Biotechnics, Hyderabad) in the deltoid region at 0, 1 and 6 months. All HIV positive patients were given 40 μg (double dose) of vaccine in each dose, while controls subjects received 20 μg (usual dose) of vaccine.

Phenotypic characterization of immune cells was done by flowcytometry on EDTA blood using FACSCAN flowcytometer with cell quest software (Becton Dickinson, Immunoflowcytometry System. San Jose, CA, USA). CD₄ ⁺ and CD₈ ⁺ lymphocytes (helper/cytotoxic) were studied using Simultest CD₄/CD₈ monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC)/phycoerythrin (PE) respectively. CD₃ ⁺ T cells (pan-T cell) and CD₁₆₊₅₆ NK (natural killer) cells were studied using Simultest CD₃ -FITC, CD₁₆₊₅₆ -PE monoclonal antibodies. Naïve T lymphocytes were studied using CD_45RA-FITC monoclonal antibodies and memory cells were enumerated using CD_45RO -PE monoclonal antibodies. The samples for flowcytometry were processed according to the method of Jackson and Warner [8].

Functional characterization of TH₁ and TH₂ type of T cells was performed on heparinised venous blood according to the method of Luty et al [9] with minor modifications. Briefly, leukocyte rich plasma was prepared from 4 ml of heparinised blood and lymphocyte count was adjusted to 3.6 × 10⁵/200 μl. Cultures were stimulated with 10% phytohemagglutinin [PHA-P (DIFCO)] in RPMI-1640 medium (Sigma) and without PHA-P (unstimulated). Cultures were incubated for 20 hrs at 37°C in an atmosphere of 5% CO₂. The samples were centrifuged at 2000 rpm for 5 minutes and the supernatant stored at -70°C until required for cytokine assay. Cytokines (IL-4, IL-10, IL-12, IFN-γ) were estimated in the culture supernatants by sandwich ELISA using reagents from Pharmingen, (USA) as per manufacturer's instructions. Briefly, culture supernatants were distributed in ELISA plates coated with corresponding anti-cytokine antibodies. Corresponding detector antibodies were used to detect cytokine anti-cytokine complexes. The reaction was developed with TMB in 0.1 M sodium acetate solutions and H₂O₂, OD was recorded at 450 nm. The concentration of the cytokines in culture supernatants were calculated from the standard curve for each cytokine plotted on a log-log paper. Functional characterization of B cells was done by estimation of sequential levels of HBsAb by ELISA.

Phenotypic markers were studied at baseline (B) and 11 days after second (D) and the third (F) dose of vaccine. Functional characterization of TH₁ and TH₂ subsets of T helper cells and levels of HBsAbs were estimated at baseline (B), before and 11 days after second (C & D) and before and 11 days after the third (E & F) dose of vaccine. The timing was selected to pick up the peak titers (Figure 1)

At base line, mean CD₄ ⁺lymphocyte counts were highest in controls followed by patients with high CD₄ counts and those with low CD₄ counts in that order (p < 0.01; Fig 2B; Table 1). After the second and the third dose of vaccination, 92% and 81% of high CD₄ group and 61% and 76% of low CD₄ group patients respectively, showed a statistically significant increase in the number of CD₄ ⁺ lymphocytes (Fig 2 and 3; Table 1).

Similarly, 65% and 85% of normal control subjects showed an increase in the number of CD₄ ⁺ lymphocytes after the second and the third dose of vaccination. The increase was however more impressive after the third dose of vaccination (Fig 3, Table 1).

At baseline, patients showed a higher number of CD₈ ⁺ lymphocytes as compared to normal subjects (p < 0.01) (Table 1). After HBV vaccination, those with high CD₄ count showed a statistically significant increase in mean CD₈ ⁺ lymphocytes after each dose of vaccination. While patients with low CD₄ count showed a statistically significant increase only after the third dose. Normal controls also showed increased mean of CD8⁺ lymphocytes after third dose of vaccination. There was a gross inversion of the CD₄/CD₈ ratio in patients before and after immunization while in controls, the ratio was one. As CD₄ ⁺ and CD₈ ⁺ lymphocytes increased after vaccination, as expected, CD₃ ⁺ lymphocytes also increased after HBV vaccination.

Basal CD16⁺56⁺ NK cell number was significantly lower in patients with low CD₄ ⁺ lymphocytes than in controls. After vaccination, patients with high CD₄ ⁺ lymphocytes showed no significant increase in number of NK cells whereas patients with low CD₄counts responded after the last dose, (p < 0.05). In controls, the NK cells increased significantly after each dose of vaccination (Table 1).

At baseline, there was no statistically significant difference in CD_45RA ⁺ cells between controls and patients with high CD₄ ⁺ lymphocytes. Patients with low CD₄ counts showed a lower number of CD_45RA ⁺ naïve cells as compared to controls and patients with high CD₄ counts (p < 0.01). After vaccination, patients with high CD₄ counts showed a significant increase in CD_45RA ⁺ naïve cells after each dose of vaccination whereas patients with low CD₄ counts and normal control subjects showed increase in CD_45RA ⁺ naïve cells after the third dose of vaccination (Fig. 4, Table 1).

Memory cells are crucial in response to any immunization. In this study, patients with high CD₄ counts showed a significantly higher number of CD_45RO ⁺ memory cells at baseline as compared to controls while patients with low CD₄ counts showed a lower number of memory cells but the difference between both the patient groups was statistically insignificant. Memory cells also increased after immunization and were higher in HIV positive patients although control subjects showed an early rise of memory cells after vaccination (Table 1).

After vaccination, 40% of patients with high CD₄ counts formed antibodies at stage C (205 ± 613 mIU/ml; p > 0.05) but 77% of patients had statistically significant levels of HBsAb at stage D i.e. 11 days after second dose (1181 ± 2912 mIU/ml, p < 0.05). Subsequently 46% of patients had transient lowering of HBsAb levels at stage E (524 ± 1478 mIU/ml p > 0.05). After third dose of vaccination at stage F, 100% of patients with high CD₄ counts showed statistically significant levels of HBsAb [8034 ± 14136 mIU/ml, p < 0.01, (Table 2)]. HBsAb, titers above 100 mIU/ml were considered significant.

In comparison, only 47% of patients who had a CD₄ count of < 200/mm³ could form HBsAb titers above 100 mlU/ml. Thirty eight percent of patients could not form antibodies at all, and 15% of patients showed HBsAb titers of < 20 mIU/ml. Thus only 47% of these patients were responders, but with significantly lower HBsAb titers (462 ± 814 mIU/ml; p < 0.5).

The response to vaccination was delayed in normal control subjects. This could be due to the different dosage schedule in patients vs. controls, the former being vaccinated with a double dose. At stage E, 85% of controls formed statistically significant levels of HBsAbs. At final stage, 90% of normal control subjects were responders and there was a striking rise of HBsAb after the last dose. Although in controls 10% were non-responders compared to patients with high CD₄ counts where 100% of patients responded, yet the mean peak titers (60000 mIU/ml) were much higher in controls than in patients with high CD₄ counts (Table 2).

Basal IL-4 levels were higher in HIV positive patients as compared to IL-4 levels in controls. Although IL-4 levels were higher in patients with high CD₄ counts than patients with low CD₄ counts yet the difference was statistically insignificant (p > 0.05) (Table 3). HBV vaccination however failed to elicit adequate IL-4 in HIV positive patients. In controls there was massive increase in IL-4 levels after the first dose of vaccination and levels were much higher at all four stages (C to F) compared to HIV positive patients (Table 3).

Basal IL-10 levels were also significantly higher in normal control subjects as compared to HIV positive patients and even after vaccination, IL-10 levels remained significantly higher in control subjects as compared to HIV positive patients (Table 3). HBV vaccination thus failed to elicit adequate IL-4 or IL-10 stimulation in HIV infected patients.

Basal IL-12 levels were almost similar in control subjects and HIV positive patients. After vaccination, all patients showed delayed but significant increase in IL-12 levels whereas in controls there was massive though transient increase in IL-12 levels (Table 3).

Baseline IFN-γ levels were much higher in control subjects as compared to HIV positive patients (p < 0.01) but the difference in basal IFN-γ levels in patients with high and low CD₄ counts was insignificant. However after vaccination, levels increased significantly in patients after 6 months. In controls, no further increase of IFN-γ levels could be registered. Yet levels were significantly higher than in patients (Table 3).

Decreased absolute CD₄ ⁺ lymphocyte number, percentage and decreased CD₄: CD₈ ratio occurs early during the course of HIV infection and are predictors of disease progression [12]. We analyzed basal CD₄ ⁺ lymphocytes and changes if any, in the number of CD₄ ⁺ lymphocytes after HBV vaccination. The basal CD₄ ⁺ cell count in Indians were found to be lower than Caucasians (743± 214). Response to any vaccine is dependant on adequate function of CD₄ ⁺ helper cells. In the present study also, response to HBV vaccination was directly correlated with CD₄ ⁺ lymphocyte number. After the second dose of HBV vaccination, 92% of patients with high CD₄ counts, showed a significant increase in the number of CD₄ ⁺ lymphocytes (peak of 1135/mm³) compared to only 61% of patients with low CD₄ counts (peak of 472/mm³). In control subjects, the increase was more impressive after the third dose and peak levels were much higher (1531/mm³). It is important to note that mean CD₄ ⁺ lymphocytes increased significantly after vaccination at each stage irrespective of the disease status.

CD₈ ⁺ lymphocytes are responsible for eliminating virus infected cells and act as a second line of defense by becoming activated and eliminating any infected cell, despite the presence of neutralizing antibodies [13]. They can inhibit viral replication by secreting T lymphocyte antiviral factor (CAF), which inhibits RNA transcription [14]. However, there is a paucity of data regarding qualitative and quantitative changes in CD₈ ⁺ lymphocytes after HBV immunization. In the present study, average number of total circulating CD₈ ⁺ lymphocytes in control subjects was 756/mm³, which is higher than the figures (500/mm³) reported in Caucasians [12]. Mean value of CD₈ ⁺ cells was much higher in HIV positive patients. After immunization CD₈ ⁺ T lymphocytes increased further in all the groups. It has earlier been documented that HIV is associated with a functional defect of CD₈ ⁺ cells. Wilson et al [15] had reported that decreased responsiveness to HBV vaccine in HIV infected subjects is associated with elevated CD₈ ⁺ CD₃₈ ⁺ HLA DR⁺ T cells. In murine studies it has been observed that repeated exposure to unrelated pathogens, as happens in HIV infected patients, may adversely affect viral specific CD₈ ⁺ lymphocytes [16].

At baseline, CD₄:CD₈ ratio in normal control subjects was approximately 1, which is also distinctly different from the values reported in the west where the ratio is close to two [12]. Our data corroborates the observation made by Sehgal et al [17] who also documented a lower CD₄ ⁺ cell number, a higher CD₈ ⁺ cell count and CD₄:CD₈ ratio of 1 in healthy North Indians.

Besides CD₄ ⁺ and CD₈ ⁺ cells, NK cells are key cells that deal with virally infected cells. Majority of peripheral blood lymphocytes that mediate NK cytotoxic activity in healthy adults express both CD₁₆ and CD₅₆ . Using these two NK cell markers, a decrease in NK cell number has been reported in HIV infected individuals [18, 19]. In the present study too, the basal mean of NK cells were low in immunosupressed patients compared to patients with high CD₄ counts and normal control subjects. After HBV vaccination, NK cells in controls increased after each dose unlike in patients with high CD₄ counts where no significant difference was observed. Patients with low CD₄ counts showed a significant increase only after the last dose of vaccination (170 vs. 293/mm³ p < 0.05). The present study thus illustrates that NK cell response is also compromised in HIV/AIDS.

In HIV infection, CD₄ ⁺ lymphocytes with the memory phenotype harbor more HIV provirus and hence are candidates for depletion by HIV cytopathic effects following activation and subsequent stimulation of viral replication [20]. Although it was initially believed that phenotypically defined memory cells were selectively decreased in HIV infected patients [21], subsequent studies showed that there is no such decrease in the cell number yet a functional impairment could be demonstrated. The exact cause of this functional impairment is still elusive [22, 23].

In the present study naïve cells were significantly lower in patients with severe disease while the number of memory cells was increased in HIV/AIDS patients. In spite of this, HBsAb levels were lower than controls thus confirming a functional defect.

In our study, HIV infected vaccinated patients failed to elicit adequate IL-4 response. IL4 and IL-10 are the two key cytokines facilitating antibody response. Klein et al [24] also demonstrated that increased IL-4 levels were seen only in early stages of HIV infection. Similarly Kid et al [25] and Romagnani et al [26] questioned the TH1/TH2 imbalance hypothesis and discussed its limitations. We also observed a significant lowering of basal mean IL-10 levels in HIV infected patients compared to controls (p < 0.01). After vaccination, patients with advanced disease showed a delayed and diminished response of IL-10. Thus both IL-4 and IL-10 responses were suboptimal in HIV positive patients, a finding at variance with that reported by Clerici and Sheara [27]. Hong et al [28] and Fakoya et al [29] also documented that TH2 cytokines were increased in HIV infected subjects. Suboptimal responses of both IL-4 and IL-10 observed with HIV patients could have accounted for weak HBsAb titers in HIV infected patients.

In the present study IFN-γ levels were much higher in controls compared to both the groups of HIV infected patients. Further, the response was delayed in patients and the IFN-γ levels could not match the levels observed in controls indicating a lack of T cell help. IL-12 levels are documented to be low in plasma of HIV infected subjects [30] but we could not document any significant difference in IL-12 levels in different groups.

Our study illustrates that HBsAb titers were highest in controls followed by seropositives and minimum in patients with AIDS. Low CD₄ ⁺ lymphocytes and low TH₂ type of cytokines i.e., IL-4, IL-10 which promote antibody function and functionally abnormal memory cells possibly accounted for low antibody titers in HIV infected patients.

Diaz et al [31] stressed that the response to HBV vaccine may be transitory in AIDS necessitating progressive monitoring of HbsAbs. In a study conducted by Walch and Morse [32], the response rate in patients with CD₄ counts of > 500/mm³ was 87% while the response rate dropped to 33% in patients with a CD₄ count between 200–500/mm³. In our study only 6/40 patients had CD₄ counts of > 500/mm³, thus the response rate of 100% in our patients with a CD₄ count between 200–500/mm³ is encouraging. These patients also gained weight and depicted a rise of CD₄ ⁺ lymphocytes. The response rate in controls receiving the conventional dose was 90%. Tedalli et al [33] recommended that patients with a CD₄ count of < 200/mm³ should be treated with HAART first and HBV vaccine given preferentially after the cut off of 200/mm³ is achieved. This, however, is not possible in resource poor countries. In an excellent comprehensive study on care of patients with chronic HBV [34], the international panel recommended that the antiHBs titers should be checked 12 weeks after immunization and booster given if the response is sub optimal. A short statement of the first European Consensus Conference on the treatment of patients with HBV and HCV reiterates that all HIV patients should be tested for viral hepatitis including those in under served areas and vaccination/treatment offered as and when necessary [35].