Neutralization of tier 1 viruses was detected in both RV14422 and Vax00323. both A3R5 and TZM-bl assays, the seroconverting points could be recognized earlier for tier 1 (15.7 weeks) and tier 2 (68.3 weeks) strains in A3R5 assay respectively. The high sensitive pseudovirus assay using more physiological target cells could serve as an alternative to the TZM-bl assay for evaluation of vaccine-induced neutralizing antibodies and recognition of the correlates of safety. KEYWORDS: HIV-1, neutralizing antibody, pseudovirus, A3R5, TZM-bl Intro It is well approved that neutralizing antibody (NAb) plays a pivotal part in most successful vaccines against infectious providers, such as polio disease, rabies disease, measles, influenza disease, human being papillomavirus.1 Since the recognition of human being immunodeficiency disease (HIV) as the pathogen of the acquired immunodeficiency syndrome (AIDS), development of the prophylactic HIV vaccine, which could induce powerful and large NAbs, has been the primary goal to fight against this epidemic. Up to now, a number of potent NAbs have been isolated from HIV-1 infected individuals2-4 and the protecting potency of them has been conformed in animal models.5,6 Unfortunately, no candidate vaccine, however, has been reported to yield this kind of NAbs. The challenge to develop an effective HIV vaccine was not just the design of appropriate immunogens, but also the establishment of standardized assays to evaluate the protecting immune responses to provide information relevant to the in vivo results and guide further modification of the immunogens. Great attempts have been invested in the development, standardization Meta-Topolin and implementation of in vitro assays for evaluating potency and breadth of NAbs against HIV-17-12. The early neutralization assays used the T-cell FANCE line-adapted (TCLA) viruses to infect cell lines, which were lately proved Meta-Topolin poorly predictive of the in vivo results. Meta-Topolin Subsequently, main isolated viruses and peripheral blood mononuclear cells (PBMCs) were utilized for in vitro neutralization assays. However, due to the genetic polymorphisms of PBMCs from different donors and the variability of the primary disease isolates, the experiment variations of intra- or inter-laboratories were quite Meta-Topolin problematic, which mainly restricted its wider applications. To circumvent variations of the checks, pseudoviruses and engeered cell lines were introduced to the neutralization assay. The pseudovirus neutralization assay based on TZM-bl cell was recommended as an optimized and validated approach to assess the sera from HIV-1 vaccines tests8, which showed a number of advantages including: high versatility of disease strains, high reproducibility, high throughput, simplicity and security of operation, and facilitation of Good Laboratory Clinical Methods validation13. The greatest concern about the manufactured cell-line centered assays was physiologic relevance and representativeness of the value for the in vivo results14. Disagreements have been reported between the results acquired between the PBMC-based and cell-line centered assays15. These discrepancies were found to be attributed to the variations of the surface molecule concentration of target cells, especially the CC chemokine receptor 5 (CCR5) quantity16, which served like a coreceptor for HIV-1 access. Cell lines with more physiological levels of CCR5 were introduced to the neutralization assay, such as the T-lymphoblastoid cell collection A3R5 with related surface CCR5 manifestation to PBMC, which used infectious molecular clones17, 18. However, when A3R5 were used as target cells for pseudovirus illness, the luciferase signal-to-noise percentage was too fragile to yield powerful results (Montefiori, personal communication). With this communication, we employed a highly efficient pseudovirus production system19 to develop a powerful pseudovirus neutralization assay based on A3R5. With all the advantages of pseudoviurs, the A3R5 assay showed significantly higher level of sensitivity than the TZM-bl assay, especially for the detection of fragile neutralization against tier 2 HIV-1 strains. Results Optimization of cell number for A3R5 neutralization assay To determine the optimal cell denseness of A3R5 assay, two NAbs (PG9 and 2F5) and two HIV-1 positive plasma samples (HB118 and BJ170) were tested against one pseudovirus (11036 from subtype B) at different cell densities.
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