Development and studies of the anti-R7V neutralizing antibody ELISA test: A new serological test for HIV seropositive patients.

Sanchez A, Gemrot F, Da Costa Castro JM.

J Immunol Methods. 2008 Mar 20;332(1-2):53-60. Epub 2008 Jan 14.

A new peptide-based ELISA test developed for the detection of anti-R7V specific antibodies in the sera of HIV seropositive patients is described. HIV virus acquires a cellular antigen at the moment the virus is released by budding, the R7V epitope. Certain patients produce anti-R7V Ab which are described as having the capacity to neutralize in vitro cell infection by HIV. Anti-R7V Ab are also detected in asymptomatic patients who have a lower likelihood of progressing to AIDS. Tested with 449 serum samples, the prevalence of anti-R7V Ab was 53.2% in HIV positive patients and 5.5% in HIV negative subjects. According to the duration of infection, the seroprevalence reaches almost 80% of non-treated long-term infected patients. Other retrospective studies were conducted on 308 HIV positive samples; the presence of anti-R7V Ab was significantly higher for 177 asymptomatic patients (64.4%) compared to the 131 symptomatic patients (35.1%). Regarding their neutralizing ability, anti-R7V antibodies were detected at the highest percentage in asymptomatic HIV-infected patients naive of treatment. Besides conventional biological parameters (CD4 and viral load), the detection of anti-R7V antibodies could be proposed to clinicians as an additional tool to manage treatment initiation and to improve the psychological state of their asymptomatic patients.

http://www.ncbi.nlm.nih.gov/pubmed/18234206?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

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Regarding their neutralizing ability, confirmed in-vitro for all variants, the anti-R7V antibodies are detected according priority to asymptomatic naïve HIV-infected patients. Initially discovered from plasma or serum belonging to so-called « long-term survivor » patients with a close to 90% correlation¹, their existence offers to clinicians and healthcare staff, a new means of investigation : for 30% to 50% of people concerned, the patients will benefit from this prognosis test to contemplate the natural non-evolution of their disease ; and the prescribing clinicians, concerned both about the quality of life of their patients and the socio-economic context, will then dispose of a wider array of elements, making it possible to justify postponing the beginning of a treatment in the absence of symptoms, or even interrupting it – be it temporary – when it seems maladjusted or difficult to respect.

The preliminary results of evaluation reporting the ELISA test efficiency², described the high specificity of the anti-R7V antibodies in case of HIV infection : based on 132 HIV sero-negative samples including possible crossing substances (HCV, HBV, HAV, EBV, HSV, Rubella, Lyme,…), a 95% specificity was calculated according to the strict validation criteria (sample declared positive if the ratio OD450 Sample / OD450 Calibrator is over 1.2). Besides, a recent retrospective study³ based on the serological follow-up (20 HIV and R7V seropositive patients), before and during HAART period as well as at its interruption, confirm the high level of specificity of these antibodies : so, naive of any treatment, 38% of the patients (8) show anti-R7V antibodies (average viral load = 186 000 copies/ml) even though after therapeutical initiation, none of them seemed to show any (average viral load < 50 copies/ml). Nevertheless, after interruption of the treatment and observed viral replication restart generating a new R7V epitope exposure, 50% of these same patients (4) showed anti-R7V antibodies again (average viral load = 121 000 copies/ml).

Regarding the data from a technical point of view, we establish that these 8 patients initially showing an average ratio (1.55) with a 47% decrease (0.82) after HAART initiation then have been showing a 48% increase (from 0.80 to 1.18) after treatment interruption. Since our kit is not a diagnosis but a prognosis tool with a large socio-economic interest, we decided to favour the sensitivity by decreasing the positive threshold to 1 leading and reducing at the same time the number of gray-zone results (positive result when ratio is over or equal to 1and borderline result when ratio is over 0.8 and strictly below 1). In these conditions (92% specificity in absence of any Rubella antecedent), we have considered 10 patients showing anti-R7V antibodies (50% of the population under study), the average ratio evoluting as described below :

- 1.46 (vs 1.55) with a 38% decrease (vs 47%) after HAART (final ratio = 0.90),

- 0.76 (vs 0.80) with a 66% increase (vs 48%) after HAART therapeutical interruption (final ratio = 1.26 – 7 out of 10 patients showing again anti-R7V antibodies, the 3 others giving borderline results).

Finally, the application of this new validation criteria on a 229 patient population (122 symptomatic under treatment^a et 107 asymptomatic without any treatment^b) leads to the following R7V serological results :

-  a : 35% vs 19% (+) ; 55% vs 55% (-) ; 10% vs 26% (gray zone)

-  b : 62.5% vs 41% (+) ; 21,5% vs 21.5% (-) ; 16% vs 37.5% (gray zone) 

As a conclusion, providing a biological and complementary element would permit to the largest number of patients to contemplate the non-evolution of their disease, which would come first to improve their psychological condition and to make them more compliant, for a more important socio-economic benefit too.

1 Galéa P. et al. 1996. Cell. Pharmacol. AIDS Sci. 3 : 311-6., 2 Haslin C. et J.C. Chermann. 2006. Spectra Bio. 155 : 82-6., 3 Cornelissen M. et al. Academic Medical Center University of Amsterdam, 2007.

Background and objective: A small number of HIV-infected patients doesn’t evolve towards AIDS. It has been demonstrated that host cells and viral factors may be involved in the non-progression of the disease. It also has been shown that the presence of specific neutralizing antibodies (Ab), targeted against the ß2-microglobulin derived peptide R7V (a cell epitope acquired by HIV during budding) is related to the patient clinical stage. After assessing the usefulness of the IVAGEN’s Anti-R7V ELISA kit, we present the final observed prevalences from asymptomatic (vs symptomatic), naïve of treatment (vs HAART) individuals. Methods : This multi-centric study is based on the cooperation of 8 hospitals (6 in Europe, 1 in Africa, 1 in Asia). Stored and new sera were tested following the product insert recommendations. Data were analyzed twice: according to 2005’s validation criteria (grey-zone : anti-R7V Ab ratio in range [0.8 – 1.2]; positive : ratio > 1.2; with a 95% specificity) and to 2007’s (grey-zone : ratio in range [0.8 – 1.0[; positive : ratio > 1.0; with a 92% specificity). Results: In Europe, a total of 299 HIV-infected patients were tested. 170 of them were asymptomatic and never treated; 129 were symptomatic and under HAART. According to 2005’s criteria, the prevalences of the anti-R7V antibodies were 48% and 19% respectively. According to 2007’s criteria, the prevalences of the anti-R7V antibodies were 65% and 35% respectively (% of grey-zone results : 29%; 14% respectively). In Africa, a total of 83 HIV-infected patients were tested. 22 of them were asymptomatic and never treated; 61 were symptomatic and also naïve of treatment. According to 2005’s criteria, the prevalences of the anti-R7V antibodies were 45.5% and 13% respectively. According to 2007’s criteria, the prevalences of the anti-R7V antibodies were 64% and 41% respectively (% of grey-zone results: 51%; 25% respectively). In Asia, a total of 32 HIV-infected patients were tested. 15 of them were asymptomatic and never treated; 17 were symptomatic and under HAART. According to 2005’s criteria, the prevalences of the anti-R7V antibodies were 73% and 29% respectively. According to 2007’s criteria, the prevalences of the anti-R7V antibodies in those selected groups were 100% and 65% respectively (% of grey-zone results : 41%; 9% respectively). Conclusions: From all geographical area, anti-R7V antibodies are significantly more often present in HIV-infected patients who remain asymptomatic (and naïve of treatment). Since the kit is not a diagnosis but a prognosis tool and in high correlation with the effects of treatment on the viral load, we decided to favour the sensitivity (consequently the overall prevalence), reducing the number of grey-zone results at the same time.