It is generally recommended that individuals with HIV infection receive influenza vaccination . Pre-highly active antiretroviral therapy (HAART) studies have demonstrated a mixed picture regarding the humoral response to influenza vaccination [2–5]. However, it has been demonstrated that patients receiving HAART can partly recover humoral responses when compared with their pre-treatment responses to vaccination . A secondary motivation for vaccination is to diminish the possibility of HIV breakthrough initiated by acute influenza infection . This may be a two-edged sword because numerous studies have demonstrated that there are patients who respond to influenza vaccination with a transient increase in their viral burden [3–5,8]. As previous studies could not demonstrate any negative effect of vaccination on the ultimate outcome of patients, the recommendation has been maintained to vaccinate against influenza.
Whether viral load elevations that occur in some patients after vaccination result in HIV-1 genomic mutations that impact on therapy is not known, even though persistent elevations after viral load elevations have been seen [9,10]. In the present study we evaluated a cohort of 34 patients with undetectable viral loads (< 200 copies/ml) on HAART, for increases in their HIV-1 plasma viral loads after influenza vaccination. HIV-1 RNA from plasma obtained from seven patients responding to vaccination with an increase in viral load was evaluated for primary reverse transcriptase (RT) and protease mutations by line probe assay (LiPA). Viral loads returned to non-detectable levels shortly after vaccination in all but two of the cases. In one of these cases, the progressive development of zidovudine mutations evolved over a year after the viral load elevation. These data taken in context with earlier studies suggest that those patients with persistent viral load elevations after influenza vaccination may develop genotypic RT mutations.
Materials and methods
During the period from October 1998 to January 1999 60 patients from the Jackson Memorial Hospital (JMH; 56) Outpatient Clinical Immunology Clinics and Miami Veterans Administration Medical Center (MVA MC; four) Outpatient Special Immunology Clinics, who were sent for influenza vaccination and had a previously undetectable viral load, consented to participate in the study as per the human experimentation guidelines of the institutional human subjects review board of the University of Miami School of Medicine. Of those consenting to participate, 34 (JMH; 31/MVAMC; three) completed the initial visit as well as the 2 and 4 week follow-up visits. All patients had been on a stable HAART regimen for at least 3 months and no patient had an active opportunistic infection. The HIV-1 stage at the time of diagnosis is shown in Table 1, and was abstracted from the available medical records. From the charts we were unable to obtain any information on whether or not a patient had B symptoms and was classified as either A or C .
Vaccination and assessment of viral load
All patients were vaccinated at their initial visit (time zero) with inactivated subvirion trivalent influenza virus vaccine, types A and B (A/Beijing/262/91 (H1N1), A/Sydney/5/97 (H3N2) and B/Beijing/184/93-like, respectively) intramuscularly as recommended by the manufacturer (Parkedale Pharmaceuticals, Inc., Rochester, MI, USA).
HIV-1 plasma viral load testing was performed in the Microbiology Laboratory of the Department of Pathology at the University of Miami/Jackson Memorial Medical Center according to manufacturer's guidelines for the Amplicor HIV-1 Monitor test (Roche Diagnostic Systems, Indianapolis, IN, USA). The limit of detection of this assay is 200 copies/ml of plasma (non-detectable < 200 copies/ml). Between 200 and 400 copies/ml is given as less than 400 copies/ml.
For patients who had an increase in their viral loads after the influenza vaccination, frozen stored plasma was evaluated by the LiPA-RT and LiPA-protease.
Patients consenting to participate in the study had blood drawn in ethylenediamine tetraacetic acid-containing tubes and plasma was collected and frozen (−80°C) immediately for viral load and T cell subset determinations at the time of vaccination (t = 0), at 2 and at 4 weeks post-vaccination. Charts were reviewed to determine viral load and medication history.
In general, Univariant analysis was used to confirm χ2 analysis. Multivariant analysis using stepwise logistic regression was used when evaluating whether independent variables were correlatively predictive.
Genotypic resistance testing for RT and protease mutations was performed by using the Innogenetics (Norcross, GA, USA) LiPA for RT and protease as described previously [12,13], and according to the manufacturers package insert. All genotypic-resistance assays were performed on the same frozen plasma samples used for the viral load determinations.
The characteristics of the 34 patients completing the full post-vaccination follow-up are shown in Table 1. The risk factors for the HIV-1-positive subjects were: 13 homosexual contacts, one bisexual contact; 14 heterosexual contacts; two injection drug users; and four with no identifiable risks. Twenty-eight patients were receiving HAART for the first time, and eight patients were on a salvage course of therapy. Of those who had a change in their plasma viral load over the course of acute monitoring, there were no outstanding characteristics (race, ethnicity, risk, age, CD4 cell count) or therapies that were predictive of a viral change after vaccination as determined by logistic analysis.
Factors influencing viral load response to influenza vaccination
The percentage of HIV-seropositive individuals who had an elevation in their viral load 2 or 4 weeks after vaccination was 21% (seven out of 34). Of the patients who responded to vaccination with an increase in their viral load, five out of 16 had CD4 cell counts greater than 500/mm3 (31%) compared with two out of 18 with CD4 cell counts less than 500/mm3 (11%). Of the eight patients on a salvage regimen three had an increase in viral load after vaccination (38%) compared with five out of 26 (19%) on first-time HAART. No patient had any acute HIV symptoms, and only patient no. 15 was on therapy for a history of cytomegalovirus retinitis, whereas the remaining six patients had no history of AIDS-defining illnesses.
Viral load and resistance profiles of those with viral load changes during the vaccination period
Plasma samples from patients with viral load elevations after vaccination were assayed for RT and protease-resistant mutations using LiPA. Table 2 is a compilation of the viral load and genotype results for each patient as a function of time measured from vaccination (zero time). Plasma from two individuals (nos. 13 and 40) was non-reactive to LiPA testing. Of the remaining five patients all had RT mutations and two of the five had protease mutations. Patient nos. 3 and 41 revealed transient mutations at 2 weeks post-vaccination (M18 4V and V/I82F, respectively). Patients nos. 41 and 81 had RT mutations that were demonstrable before and after vaccination.
Patients nos. 12 and 15 revealed elevated viral burdens that occurred after vaccination and persisted. Patient no. 12 demonstrated the development of multiple zidovudine mutations after inoculation (Table 2). At 19 weeks after inoculation the patient's primary mutation pattern revealed a K70R mutation, with the development of the T215F mutation at 53 weeks. The D30N protease mutation seen in Table 2 for patient no. 12 developed at a time before vaccination. Patient no. 12 was a compliant patient with well-controlled hypertension and diabetes. He had home healthcare personnel who dispensed medication. Patient no. 15, who had a persistent elevation in viral load after vaccination, had his medication subsequently changed to zidovidine, lamivudine, indinavir, and ritonavir instead of a regimen containing nevirapine. The patient's viral loads subsequently fell below 400 copies/ml. Multiple attempts, in different laboratories, to evaluate sequence genotyping at time-points when the patient had viral loads of 1000 copies/ml or greater were unsuccessful. All of the patient's genotypic mutations were consistent with their current or past therapies.
To achieve complete suppression of viral replication in clinical practice is difficult [14–16]. Therefore, it becomes important in the clinical population to evaluate the effect of events and interventions that could significantly affect viral load levels and impact on ultimately successful interventions. Some individuals with HIV disease respond to an influenza vaccination with a transient increase in their viral load [3–5,8]. Studies evaluating the effect of vaccination on viral burden for individuals on HAART with undetectable viral loads are limited [9,10]. An inability to control viral load to less than 50 copies/ml seems to result in significant viral load elevations after influenza vaccination , and in some cases results in a persistent elevation. This is distinct from the transient small elevations that occur in individuals with suppressed viral burden (< 50 copies/ml) that may [17,18] or may not [19–21] result in drug-resistant mutations. In our study, we evaluated the plasma viral load response after influenza vaccination of patients with an undetectable viral load (< 200 copies/ml) at the time of inoculation for primary RT and protease drug-resistant mutations by LiPA. This technology was reliable and sensitive  but did, on occasion, fail to amplify DNA from patients with high viral burdens.
Transient primary mutations were noted in two patients after inoculation. Whether these primary mutations represent new mutations or the expression of an archived mutation is not known. Data from studies using the heteroduplex tracking assay [22,23] have shown that evanescent viral species occur while on suboptimal therapy, and may arise from viral reservoirs harboring distinct variants. It is also possible that at low viral copy numbers a selection bias during amplification may exist, reflecting a complex pool of viral species.
Two patients had sustained elevations in viral burden after influenza vaccination. One patient had no RT or protease mutations. This patient was on a regime containing nevirapine. Genetic sequencing of plasma RNA of historical samples, post-vaccination samples, and current samples all failed to yield amplifiable DNA in order to evaluate whether a non-nucleoside reverse transcriptase inhibitor mutation occurred. As the LiPA-protease and LiPA-RT only display primary mutations for those regions and do not evaluate non-nucleoside reverse transcriptase inhibitor mutations we cannot comment on whether medication failure was caused by the loss of this drug class. The second patient (no. 12) had a slow return to undetectable viral load with no change in therapy. After vaccination, many new zidovudine mutations were noted and accumulated over time from vaccination. The sequence of evolving mutations and the timing of each mutation suggests that these are new zidovudine mutations instead of archived variants .
The data in this paper are important because they contribute to other studies that evaluate the effect of vaccination on viral burden for patients with undetectable viral loads. To date, this is the only such study that looks at the genotypic profiles as a function of time from vaccination, and is able to show the development of an RT mutation after vaccination, with viral load persistence. Our work further suggests that the evaluation of morbidity and mortality evaluated over 1–2 years  may not be sufficient to demonstrate a negative outcome because of the long time-course of evolving mutations. The percentage of individuals elevating their viral load after influenza vaccination is not small, and as such deserves further evaluation with regard to whether such challenges provide a platform for enhancing viral evolution and the establishment of new archived viral species.
The authors would like to thank Ms S. Young for the chart abstractions.
1. Centers for Disease Control and Prevention. Prevention and control of influenza: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR 1999, 48 (no.RR-4): 1–22.
2. Tasker SA, O'Brien WA, Treanor JJ. et al
. Effect of influenza vaccination in HIV-infected adults: a double-blind placebo-controlled trial. Vaccine 1998, 16: 1039–1042.
3. Staprans SI, Hamilton BL, Follansbee SE. et al
. Activation of virus replication after vaccination of HIV-1-infected individuals. J Exp Med 1995, 182: 1727–1737.
4. Rosok B, Voltersvik P, Bjerknes R, Axelsson M, Haaheim LR, Asjo B. Dynamics of HIV-1 replication following influenza vaccination of HIV+
individuals. Clin Exp Immunol 1996, 104: 203–207.
5. Fuller JD, Craven DE, Steger KA, Cox N, Heeren TC, Chernoff D. Influenza vaccination of human immunodeficiency virus (HIV)-infected adults: impact on plasma levels of HIV type 1 RNA and determinants of antibody response. Clin Infect Dis 1999, 28: 541–547.
6. Kroon FP, Rimmelzwaan GF, Roos MTL. et al
. Restored humoral immune response to influenza vaccination in HIV-infected adults treated with highly active antiretroviral therapy. AIDS 1998, 12: F217–F223.
7. Couch RB, Englund JA, Whimbey E. Respiratory viral infections in immunocompetent and immunocompromised persons. Am J Med 1997, 102: 2–9.
8. Ho DD. HIV-1 viraemia and influenza. Lancet 1992, 339: 1549.1549.
9. Gunthard HF, Wong JK, Spina CA. et al
. Effect of influenza vaccination on viral replication and immune response in persons infected with human immunodeficiency virus receiving potent antiretroviral therapy. J Infect Dis 2000, 181: 522–531.
10. D'Vorah H, Thompson CE, Salvato PD. Effect of influenza vaccination on viral loads in HIV patients.
In:The 12th World AIDS Conference
. Geneva, June 1998 [Abstract 32155].
11. Centers for Disease Control and Prevention. 1993 Revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR 1992, 41 (no. RR-17): 1–19.
12. Stuyver L, Wyseur A, Rombout A. et al
. Line probe assay for rapid detection of drug-selected mutations in the human iImmunodeficiency virus type 1 reverse transcriptase gene. Antimicrob Agents Chemother 1997, 41: 284–291.
13. Servais J, Lambert C, Fontaine E. et al
. Comparison of DNA sequencing and a line probe assay for detection of human immunodeficiency virus type 1 drug resistance mutations in patients failing highly active antiretroviral therapy. J Clin Microbiol 2001, 39: 454–459.
14. Ledergerber B, Egger M, Opravil M. et al
. Clinical progression and virological failure on highly active antiretroviral therapy in HIV-1 patients: a prospective cohort study. Swiss HIV Cohort Study.
Lancet 1999, 353: 863–868.
15. Lucas GM, Chaisson RE, Moore RD. Highly active antiretroviral therapy in a large urban clinic: risk factors for virologic failure and adverse drug reactions. Ann Intern Med 1999, 131: 81–87.
16. Deeks SG, Barbour JD, Martin JN, Swanson MS, Grant RM. Sustained CD4+ T cell response after virologic failure of protease inhibitor-based regimens in patients with human immunodeficiency virus infection. J Infect Dis 2000, 181: 946–953.
17. Martinez-Picado J, DePasquale MP, Kartsonis N. et al
. Antiretroviral resistance during successful therapy of HIV type 1 infection. Proc Natl Acad Sci U S A 2000, 97: 10948–10953.
18. Gunthard HF, Wong JK, Ignacio CC. et al
. Human immunodeficiency virus replication and genotypic resistance in blood and lymph nodes after a year of point antiretroviral therapy. J Virol 1998, 72: 2422–2428.
19. Havlir DV, Bassett R, Levitan D. et al
. Prevalence and predictive value of intermittent viremia with combination HIV therapy. JAMA 2001, 286: 171–179.
20. Ramratnam B, Mittler JE, Zhang L. et al
. The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy. Nat Med 2000, 6: 82–85.
21. Hermankova M, Ray SC, Ruff C. et al
. HIV-1 drug resistance profiles in children and adults with viral load of < 50 copies/ml receiving combination therapy. JAMA 2001, 286: 196–207.
22. Delwart EL, Pan H, Neumann A, Markowitz M. Rapid, transient changes at the env
locus of plasma human immunodeficiency virus type 1 populations during the emergence of protease inhibitor resistance. J Virol 1998, 72: 2416–2421.
23. Liu SL, Schacker T, Musey L. et al
. Divergent patterns of progression to AIDS after infection from the same source: human immunodeficiency virus type 1 evolution and antiviral responses. J Virol 1997, 71: 4284–4295.
24. Boucher CA, O'Sullivan E, Mulder JW. et al
. Ordered appearance of zidovudine resistance mutations during treatment of 18 human immunodeficiency virus-positive subjects
. J Infect Dis 1992, 165: 105–110.
25. Sullivan PS, Hanson DL, Dworkin MS, Jones JL, Ward JW. Effect of influenza vaccination on disease progression among HIV-infected persons. Adult and Adolescent Spectrum of HIV Disease Investigators.
AIDS 2000, 14: 2781–2785.