A high human papilloma virus (HPV) prevalence and an augmented incidence of infections with oncogenic HPV types are seen in HIV patients . CD4+ levels likely influence the outcome of HPV infection in these patients because the alterations in cell-mediated immunity (CMI) associated with HIV disease favour its persistence . From a clinical standpoint, even if antiretroviral therapy (ART) can result in a very satisfactory degree of immune reconstitution, HPV-associated low-grade squamous intraepithelial lesions persist longer and are more likely to evolve into high-grade lesions in HIV patients . Importantly, HPV infection associated complications are not only detected in HIV-infected women, as significant increases in penile and anal cancer are seen in HIV-infected men .
One of the approved HPV preventive vaccines (Gardasil) is a quadrivalent preparation that contains HPV types 16 and 18 virus-like particles, stimulates high titres of neutralizing antibodies and is extremely effective in preventing precancerous lesions of the cervix, vulva and vagina, as well as genital warts in immunocompetent hosts . We recently showed that Gardasil elicits strong humoral responses in HIV-infected adolescents and young adults ; here, we analysed CMI responses to such vaccine.
Forty-six HIV-infected adolescents and young adults were enrolled in a longitudinal, prospective, nonrandomized, controlled, open-label clinical study to assess the long-term immunogenicity of the HPV vaccine Gardasil used thrice (baseline, 8 and 24 weeks) intramuscularly. The characterization of the individuals enrolled in the study as well as the detailed vaccinal schedule was recently described .
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and incubated in the presence/absence of purified recombinant HPV-6/11/16/18 L1 proteins (5 μg/ml; Meridian Life Science, Memphis, Tennessee, USA). Anti-CD28 antibody (R&D Systems, Minneapolis, Minnesota, USA) was added during incubation (1 μg/well). For cytokine analyses, 10 μg/ml Brefeldin A (Sigma-Aldrich, St Louis, Missouri, USA) was added during the last 6 h of stimulation.
Lymphocyte subsets were evaluated by fluorescence-activated cell sorting using 50 μl ethylenediaminetetraacetic acid peripheral blood incubated for 30 min at 4°C with fluorochrome-labelled mAbs. CD4 and CD8 T lymphocyte maturation pathway was analysed by evaluating the expression of CCR7 and CD45RA as previously reported [7,8]. PBMCs were stained with anti-CD4 and anti-CD8 mAbs, washed and fixed in Reagent A solution (FIX & PERM Cell Permeabilization kit; Caltag Laboratories, Burlingame, California, USA). Cells were then resuspended in reagent B (FIX & PERM) with protein-specific mAbs [7,8].
Cytometric analyses were performed using a FC500 flow cytometer (Beckman-Coulter, Milan, Italy) equipped with a double 15-mW argon ion laser operating at 456 and 488 interfaced with Intercorp computer as previously described [7,8].
Comparisons between the different groups were made using a two-tailed t-test. Statistical analysis was performed using the SPSS statistical package (SPSS, Chicago, Illinois, USA).
No significant safety issues were identified. CD4+ T lymphocyte count remained stable and HIV RNA load was undetectable during the study period in most individuals .
Naive (CCR7+/CD45RA+) and central memory (CM) (CCR7+/CD45RA−) CD4+ and CD8+ T lymphocytes were increased, whereas terminally differentiated (TD) (CCR7−/CD45RA+) CD4+ and CD8+ T lymphocytes were reduced at the 12 weeks timepoint. At 28 weeks, naive and CM CD4+ and CD8+ T lymphocytes were diminished, whereas effector memory (EM) (CCR7−/CD45RA−) and TD CD4+ and CD8+ T lymphocytes were augmented in the same individuals (Fig. 1a). No differences were detected between HIV-infected individuals and heathy controls (HC) with the exception of EM CD4+ T lymphocytes that were reduced at week 28 in patients alone.
Activated CD4+ (CD25+/HLA-DRII+) and CD8+ (CD25+/HLA-DRII+) T lymphocytes were also increased in HIV-infected and uninfected individuals at week 12 and 28 compared with baseline (Fig. 1b). Both these populations were significantly higher in HIV-infected individuals at all time points. Finally, CD8+/CD38+/CD45RO+ cells remained constant throughout the study period (Fig. 1b).
HPV-specific interleukin (IL)-2- and IL-2/interferon-gamma (IFNγ)-secreting CD4+ T lymphocytes as well IFNγ-producing CD8+ T lymphocytes were significantly augmented as well in both groups of individuals at weeks 12 and 28 compared with baseline (Fig. 1c). CD8+ T-cell (CTL) effector mechanisms were also potentiated, as both tumour necrosis factor-alpha (TNF-α) production by HPV-stimulated CD8+ T lymphocytes and perforin and granzyme B expressing CD8+ T lymphocytes were increased by vaccination (Fig. 1d).
Treatment options for HPV-associated diseases remain limited, but efficient vaccines are nowadays available . In HIV-infected patients, the American Council of Immunization Practices recommends HPV vaccination for women and men aged 11–26 years. Although few efficacy data are available in HIV-infected individuals, this recommendation is based on clinical evidence that the quadrivalent HPV vaccine (QHPV) is well tolerated and highly immunogenic in perinatally HIV-infected children  and in HIV-positive men .
We previously showed that the standard QHPV three-dose regimen generates long-lasting antibody responses in HIV-infected, ART-treated adolescents and young adults with undetectable viremia and effective CD4+ T-cell recovery . The data reported in the present study indicate that a significant CMI response, resulting in a functional activation of the T-cell compartment that is probably pivotal in achieving both the preventive and the therapeutic effect of such vaccine , is also observed in these individuals. Notably, our data demonstrate that the CMI immune responses elicited by QHVP are qualitatively similar in HIV-infected and HIV-uninfected individuals.
At the end of study period, an important increase in late stage differentiated CD4+ and CD8+ subsets was detected. These data are expected on the basis of the lymphocytes physiological lineage relationship , and are important considering that late stage cells optimally mediate effector mechanisms against pathogens . Activated T lymphocytes were increased as well by the vaccine protocol, and QHPV also resulted in a robust CMI response characterized by Th1-type cytokines and cytotoxic CTL responses. This is biologically relevant, as CTLs play a critical role in clearing established HPV genital infection . CTLs are stimulated by Th1-type cytokines and can kill targets via granule-independent (TNF-α) and granule-dependent (perforin and granzymes) mechanisms. QHPV generated HPV-specific CTLs in all vaccines and augmented the release of TNF-α, perforin and granzyme B by HPV-specific CD8+ T lymphocytes, indicating that both CTL-mediated effector mechanisms are potentiated by this vaccine.
HIV infection results in immune alteration [16–18] that are properly, although not totally, reversed by ART . Given this consideration and the great incidence and prevalence of HPV in HIV-infected patients [1,20], vaccine formulations that are well tolerated and effective in this population are needed. The data reported in the present study showing that three-dose QHPV vaccination induces robust CMI responses in HIV-infected adolescents and young adults in the context of a satisfactory immunovirologic response to ART support the recommendation to vaccinate HIV-infected individuals.
This work was supported by by Istituto Superiore di Sanità ‘Programma Nazionale di Ricerca sull’AIDS’ and Ricerca Finalizzata and Ricerca Corrente (Italian Ministry of Health).
V.R. and F.C. performed immunological analyses and contributed in result interpretation.
V.G., F.P., V.F., C.M. and S.P. enrolled patients and controls and administrated HPV vaccine.
G.V.Z., M.C. and D.T. designed, executed and interpreted the study, and wrote the manuscript
Conflicts of interest
There are no conflicts of interest.
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