Specificity of anti-human leukocyte antigen antibody responses after immunization with Remune, an inactivated HIV-1 vaccine
Page, Marka; Ojugo, Agathaa; Imami, Nesrinab; Hardy, Garethb; Gotch, Francesb; Almond, Neila
aDivision of Retrovirology, National Institute for Biological Standards and Control, Potters Bar EN6 3QG, UK
bDepartment of Immunology, Imperial College, Chelsea and Westminster Hospital, London SW10 9NH, UK.
Received 13 July, 2006
Revised 2 October, 2006
Accepted 30 October, 2006
Antibody responses against human leukocyte antigen (HLA) classes I and II were detected in HIV-1 infected individuals who received a fixed inactivated HIV-1 (Remune) immunotherapy. The response was specific for HLA-B62 and HLA-DR4 concordant with the host cell line, HUT-78, used in vaccine production. These responses were not detected in HLA-B62 and HLA-DR4-positive individuals indicating that immunotherapy did not break tolerance to self-antigens.
Remune is an inactivated HIV-1 vaccine currently under clinical development for the immunotherapeutic treatment of HIV-1-positive individuals undergoing antiretroviral therapy . This immunotherapy is designed to elicit antiviral immunity that will provide enhanced control of virus infection and consequently delay disease progression. It was recently reported that the immunotherapeutic administration of Remune elicits anti-human leukocyte antigen (HLA) antibodies in HIV-1-positive individuals . These preliminary data corroborate previous observations that fixed inactivated SIV vaccines prepared in human T-cell lines were capable of eliciting potent anti-HLA responses in macaques [3–6]. Generating such responses in a xenogeneic setting would be anticipated; however, in an allotypic setting it raises the potential of autoimmunity. This study was designed to investigate these anti-HLA responses in greater detail.
Sera were collected from 35 HIV-1-positive male patients enrolled into a phase 1, randomized, controlled study conducted at the Chelsea and Westminster Hospital, London, UK . All patients were initiated onto HAART for 17 weeks before randomization into four groups. Group A were maintained on HAART for a further 47 weeks; group B were maintained on HAART and received treatment with 5 × 106 units of IL-2 (Proleukin) subcutaneously, twice a day at least 8 h apart; group C were maintained on HAART and received treatment with IL-2 as above, plus Remune (100 μg) administered intramuscularly with Freund's incomplete adjuvant at weeks 17, 29, 41 and 53; and group D were maintained on HAART and received treatment with Remune as above. Serum samples were collected before the initiation of HAART, before randomization (week 17) and at weeks 41, 53 and 65.
HIV-1 immunogen (Remune) is derived from a Zairian HIV-1 isolate (HZ321), an intersubtype recombinant of clade A envelope and clade G Gag  propagated on the human T lymphoblastoid cell line (HUT-78) . Virions in the extracellular supernatant of this culture were purified by ultrafiltration and ion-exchange chromatography. The antigen is then inactivated through a sequential application of β-propiolactone and 60Co irradiation . During this process the envelope protein (gp120) is removed.
Anti-HLA antibodies were characterized by Luminex using Lifematch antigen bead sets. HLA haplotypes of patients were assessed by polymerase chain reaction–sequence-specific primer as described previously .
No anti-HLA antibody responses were detected before the initiation of immunotherapy. Five patients were identified as having anti-class I responses after treatment, including one patient from group B who received HAART and IL-2 (F577), one patient from group C who received HAART, IL-2 and Remune (J565), and three patients from group D who received HAART and Remune (O152, R362 and S433). For the class I responses, bead set numbers 105 (A2, B49, B62, Bw4, Bw6, Cw3, Cw7), 112 (A25, A68, B51, B62, Bw4, Bw6, Cw15, Cw3), 137 (A2, A26, B62, Bw6, Cw3, Cw7), 156 (A11, A29, B56, B62, Bw6, Cw1, Cw3), 159 (A1, A11, B75, B8, Bw7, Cw7, Cw8) and 183 (A3, A33, B42, B63, Bw4, Bw6, Cw17, Cw5) were consistently reactive (mean fluorescence intensity > 5000) with responding patient sera. In four of the bead sets (105, 112, 156, and 159) HLA B62 was the only common antigen present (Table 1).
Specific anti-HLA class II responses were detected consistently (mean fluorescence intensity > 20 000) against bead sets 126 (DR1, DR4, DR53, DQ5, DQ7), 128 (DR4, DR53, DQ7, DQ8), 134 (DR14, DR4, DR52, DR53, DQ5, DQ8) and 160 (DR4, DR53, DQ5, DQ8) by selected sera. A total of 13 patients were identified as responders. These included three patients from group B treated with HAART and IL-2 (B231, 0992 and F577), five patients from group C who were treated with HAART, IL-2 and Remune (H140, F652, O232, S566 and C425), and five patients from group D treated with HAART and Remune (S433, W425, B318, L685 and K235). HLA-DR4 is common to the four bead sets against which seroreactivity was detected (Table 1).
The generation of specific anti-HLA antibody after immunotherapy raises concern as to whether they represent auto-immune responses. Therefore we determined the HLA expression profiles of each patient in groups C and D and responding patients in group B (Table 1). A total of seven patients of those treated with Remune were demonstrated to express HLA-B62 or HLA DR4 alleles. None of these patients produced detectable antibody responses against their own alleles, which is reassuring with regard to concerns over the induction of autoantibodies.
This paper confirms the observation of Fernandez-Cruz et al.  that patients treated with Remune, a whole inactivated HIV-1 vaccine, elicit antibodies against HLA class I and class II. Although the bead sets used to detect anti-HLA antibodies contain multiple specificities in each well, it is possible to deconvolute the responses detected against HLA to a single allele. As a result, antibodies against both class I (HLA-B62) and class II (HLA-DR4) were detected specifically. The specificities of the responses were consistent with the allelic phenotype of the HUT-78 cell line (A1, B62, Cw3, DR4 and DQ3 ) used to propagate the vaccine virus.
The higher response rate against HLA-DR4 compared with that against HLA-B62 is likely to reflect a dose–response effect because of the reported higher content of HLA-DR (11.7%) compared with class I (0.7%) on the vaccine particle .
It was surprising that similar anti-HLA antibody responses were detected in a small proportion of patients who were not vaccinated with Remune (group B) but were treated with IL-2. However, IL-2 is an immunostimulatory component and may have elicited non-specific but HLA crossreactive antibody responses.
The observations in this study may re-invigorate research into the potential of fixed inactivated HIV-1 as a prophylactic AIDS vaccine. Over 15 years ago, it was demonstrated that protection conferred by fixed inactivated SIV vaccines correlated with potent serological responses against host cell components incorporated into virions . Subsequent studies indicated that immunization with HLA class I and class II derived from the human T-cell lines used in vaccine preparations could reproduce this protection [4–6]. There is, however, only a single report of similar protection being obtained in an allo-immune setting . Further studies are needed to examine whether the anti-HLA antibodies generated by the administration of Remune possess antiviral properties.
Sponsorship: N.I. and F.G. are supported by funding from the MRC (grant no. G0501957) and AVIP EU Programme (grant no. LSHP-CT-2004-503487). A.O. was supported by funding from the MRC (grant no. G9025730).
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