The emergence of a new distinct subtype of the influenza virus has the potential to produce global pandemics that can lead to significant morbidity and mortality if the new strain is particularly pathogenic. During the spring of 2009, a novel influenza A H1N1 virus was identified, initially in Mexico and the USA and spreading quickly to the rest of the world. In June 2009, the WHO declared a flu pandemic, only the fourth of the last century .
Some immunosuppressed individuals are at an increased risk of developing complications after influenza infections . Although there are scant data supporting that patients with HIV infection have a higher influenza case rate [3,4], several studies have demonstrated that HIV-infected patients are more likely to have severe or prolonged influenza infections [5,6]. The widespread use of HAART has decreased the rate of complications of influenza in HIV-infected individuals, but has not brought them down to the rate of the uninfected population . As a consequence of this, treatment guidelines recommend yearly influenza vaccination for all patients with HIV infection [8,9]. The transient increases in HIV replication observed after influenza vaccination has not translated into long-term deleterious effects [10–15]. Multiple studies have demonstrated that antibody responses after influenza immunization among HIV-infected individuals are poorer than in the general population, although this finding has not been consistent across all studies [16–23]. The main predictors of vaccine responses in this population have been the CD4 cell counts  and the presence of HIV viremia [23,25]. Except for a few studies [24,26], almost all trials evaluating immunological responses to the influenza vaccine among HIV-positive individuals have looked at serological responses rather than the incidence of clinical influenza .
Several epidemiologic studies describing risk factors for severe H1N1 infection do not separate HIV infection from other immunosuppressive states, they have shown consistently that individuals with underlying immunosuppression are more prone to both infection and more severe cases of influenza [28,29]. This has been particularly worrisome in South Africa, where 53% of the deaths from H1N1 had underlying HIV infection , raising concerns of the potential lethality of this infection in these patients, particularly in the developing world. Mexico's data suggest that patients with CD4 cell counts less than 200 cells/μl or an opportunistic infection are at particular risk for complications requiring inpatient care [relative risk (RR) 24.5] and mechanical ventilation (RR 19.7) . However, in the developed world, the rate of infection and complications after H1N1 infection is similar or lower in HIV-infected individuals when compared with HIV-negative individuals [32,33].
We conducted this study to evaluate the immunogenicity, safety and tolerability of one of the currently approved and recommended H1N1 vaccinations for HIV-infected individuals.
We evaluated the immunogenicity, safety and tolerability of the recommended single intramuscular 15 μg dose of the monovalent, unadjuvanted, inactivated, split virus H1N1 vaccine (Novartis, Basel, Switzerland). Each participant had baseline studies performed at the time of enrollment followed by the intramuscular administration in one of the deltoid muscles of the 2009 H1N1 influenza vaccine (0.5 ml), followed by two phone calls and serological response evaluations completed 21–28 days after vaccination. Our hypothesis was that the response to the new H1N1 vaccine would be compromised in patients with HIV infection.
We included HIV-infected individuals, older than 18 years of age, who had an indication to receive the H1N1 vaccine. We excluded individuals with a known allergy to eggs or other components of the vaccine, a history of severe reactions to previous immunization with seasonal flu, known cases of H1N1 influenza during the spring of 2009 or previous recipients of the novel H1N1 vaccine. We also excluded recipients of other licensed live vaccines within 4 weeks or inactivated vaccines within 1 week of study entry. Patients receiving experimental treatments (except participants of phase III antiretroviral trials), systemic chemotherapy for the previous 36 months, steroids and other immunomodulators, or with a history of Guillain–Barré syndrome were also excluded.
Sequential clinic patients who agreed to participate were enrolled. The study was conducted at the MacGregor Clinic of the Hospital of the University of Pennsylvania in Philadelphia, Pennsylvania, USA, between the months of November and January 2009–2010. All patients signed an informed consent. The study was approved by the University of Pennsylvania institutional review board and registered in clinical trials.gov #NCT01111162.
The primary immunogenicity endpoint of the study was the proportion of participants with antibody titers equal or greater than 1: 40 on the hemagglutination-inhibition (HAI) assay. The primary safety endpoint was the frequency, duration and intensity of adverse events after vaccination [solicited, local and systemic, using the HIV Vaccine Trials Network (HVTN) questionnaire]. Seroconversion was defined as a four-fold increase from baseline titers.
We collected local and systemic adverse events using a modified HIV Vaccine Trials Network (HVTN) questionnaire, which has been validated by the HVTN. We used the standard AIDS Clinical Trials Group grading scale to evaluate adverse events.
We measured anti-HAI assay antibody titers at baseline and at week 3 (+7 days) after immunization, using the HAI assay against the H1N1 (A/California/04/2009) strain (Bioqual, Inc., Rockville, Maryland, USA).
Hemagglutination inhibition assay
Sera were treated with receptor-destroying enzyme (RDE) by diluting one part serum with three parts enzyme and incubated overnight at 37°C in a water bath. The enzyme was inactivated by 30-min incubation at 56°C followed by adsorption to red blood cells (RBCs) and addition of phosphate buffer saline for a final dilution of 1/10. HAI assays were performed in V-bottom 96-well plates using four hemagglutinating units (HAU) of virus and 0.5% turkey RBCs.
A total of 120 participants were enrolled. We anticipated that up to 20% of the participants would not come for the second blood draw at 21–28 days. Finding of no grade 3 or grade 4 adverse events in a total evaluable sample of 100 participants would provide 95% confidence that the rate in the population from which the sample was drawn is no greater than 3%. Finding of 75 immunological responders in the total sample of 100 participants would provide 95% confidence that the rate of response in the population from which the sample was drawn was no lower than 67% (PASS 2008; NCSS, LLC., Kaysville, Utah, USA). Summary statistics for continuous variables are presented as median (first quartile–third quartile). Comparison between treatment arms was carried out using a Mann–Whitney U test for continuous variables, and a Fisher's exact test or Pearson's χ2 test for categorical factors. A logistic regression model was constructed to evaluate current and nadir CD4 cell counts, viral load (undetectable or not), age and race as predictors of response or not to the H1N1 vaccine. The SPSS statistical package (version 17.0; SPSS, Inc., Chicago, Illinois, USA) was used for the analysis of the trial data.
From 20 November to 17 December 2009, we enrolled 120 participants. All vaccinated participants completed the second visit at 21–28 days after immunization. All 120 participants are included in the immunogenicity and safety analysis.
The participants' baseline characteristics are summarized in Table 1. Participants were mostly men (85%) and African–American (68%). All but one were receiving antiretroviral therapy (ART), and most of them had an HIV RNA viral load below 400 copies/ml (92%, 84% of them below the limit of quantification using the ultrasensitive assay) for a median of 26 months. The median nadir CD4 cells count of patients was 131 cells/μl, and the current CD4 cells count was 502 cells/μl.
Thirty of the 120 (25%) participants had antibody HAI assay titers equal or greater than 1: 40 at baseline. At week 3, 69% of participants achieved antibody levels equal or above 1: 40 (Fig. 1). Among the 90 participants without evidence of previous exposure to H1N1, only 61% [95% confidence interval (CI) 51–71] developed protective titers by week 3 of the study. Nonresponders had lower current (394 vs. 497 cells/μl) and nadir CD4 cell counts (112 vs. 153 cells/μl) and had an undetectable HIV viral load for a shorter period of time than responders (19 vs. 28 months), although those differences did not reach statistical significance (Table 2). Only four out of nine participants with detectable HIV viral load at baseline (and no evidence of prior infection) developed protective antibody titers. Race and age did not differ among responders and nonresponders to the vaccine. The rate of seroconversion (a four-fold increase from baseline titers) was 56% (95% CI 47–65). Among participants with baseline titers below 1: 40, the rate of seroconversion was 53% (95% CI 34–72), and for those with baseline titers greater than or equal 1: 40, it was 57% (95% CI 46–67) (P = NS).
In a logistic regression model that evaluated current and nadir CD4 cell counts, viral load, sex, age and race as potential predictors, only current CD4 cells count (P = 0.019) was associated independently with a vaccine response. Among participants in the lowest quartile of current CD4 cell counts (31–306 cells/μl), only 43% had seroprotection at the end of the study compared with approximately 60% among participants in the three highest quartiles (Fig. 2).
The vaccine was well tolerated with grade 1 local reactions at the site of injection, observed in 18% of the participants. There were no serious adverse events. Figure 3 summarizes adverse events observed. The most frequent local adverse events were pain and tenderness at the injection site. The most frequent systemic side effect was malaise followed by headache and myalgias.
H1N1 vaccination has been clearly effective in the adult healthy population. A single 15 μg dose of 2009 H1N1 vaccine was immunogenic in adults, with mild-to-moderate vaccine-associated reactions. More than 95% of the recipients developed protective antibody titers .
Our study shows that independently of virological control and CD4 recovery, up to 40% of HIV-positive individuals are not seroprotected after vaccination. The presence of other underlying chronic diseases, medication use, poor nutrition, irreversible damage to the immune system and immunosenescence, likely all play a role in decreased vaccine responsiveness in spite of the successful treatment of the HIV infection.
The implications of this research are immediate for next year's influenza vaccination campaign, which will substitute the H1N1 component of the trivalent vaccine (the only one currently approved for use in HIV-infected individuals) with the 2009 H1N1 pandemic strain. Our results suggest that if that vaccine is used at the currently recommended dose, a significant proportion of the individuals will remain vulnerable to influenza. Furthermore, although some studies suggest otherwise , a 1: 40 titer may not be fully protective in immune-compromised individuals.
What strategies can be used to improve these immunological responses? Higher dose of the antigen has been associated with higher antibody titers in many studies and can improve the immunogenicity of the influenza vaccine in poorly responsive populations such as the elderly . This strategy in adult HIV-infected individuals is currently being evaluated in two National Institutes of Health sponsored studies (clinical trials.gov #NCT00996970 and #NCT00992433). Another strategy to improve immunogenicity in the population of HIV-infected individuals is the use of adjuvants that can increase the responsiveness to this and other vaccines . During the 2010 Conference of Retrovirus and Opportunistic infections in San Francisco, two studies using the same AS03A-adjuvanted H1N1v vaccine showed divergent results, one positive  and one negative ; however, the use of adjuvanted influenza vaccines have not been approved in the United States. The use of the live virus vaccine may improve responses, but recent studies in adults suggest that this vaccine is less immunogenic in adults than the trivalent vaccine, particularly in those individuals with some degree of preexisting immunity , and this live attenuated vaccine is not approved for use in HIV-infected individuals in spite of some promising preliminary results [40,41].
In our study, current CD4 cells count, and not nadir CD4 cells count, was the strongest predictor of vaccine response. We had a very small proportion of patients with detectable viremia and, therefore, our study was underpowered to evaluate the effects of persistent viremia in vaccine responsiveness, which in other studies have been associated with vaccine's responsiveness . Thanks to the advances on ART, the population of patients with detectable viremia in HIV-infected patients in care is dwindling; however, they probably constitute a population with high risk for poor responses to vaccination and another reason to the growing number of arguments to consider earlier treatment of HIV infection.
Because of the advances of antiretroviral treatment and the increased survival of patients with HIV infection, the prevalence of HIV infection in the population will continue to grow both in the developed and the developing world. Alternative vaccines, dosing, adjuvants or schedule strategies are needed to achieve effective immunization of this vulnerable population. The 2009 H1N1 infection, though less severe than originally feared, could serve as a good model for future, potentially more deadly pandemics. Lessons learned from this epidemic can, and should, be applied in the future and help in the design and development of more effective vaccines.
The study was supported in part by the National Institute of Allergy and Infectious Diseases grant #U01-AI069467 and the Center for AIDS Research grant #P30-AI045008 to the University of Pennsylvania.
All authors participated in the design, implementation, analysis and interpretation of the study.
There are no conflicts of interest.
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