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The ability to readily elicit broadly neutralizing antibodies to HIV-1 remains elusive. We and others have hypothesized that interaction of the viral envelope glycoprotein (Env, gp120-gp41) with its receptor molecules could enhance the exposure of conserved epitopes that may facilitate the elicitation of broadly neutralizing antibodies. The Env-CD4-coreceptor complexes mediate HIV-1 entry into cells and serve as a major target for inhibitors of this process. To begin to evaluate their potential also as vaccine immunogens we produced relatively large amounts of complexes of purified recombinant soluble truncated Env, gp14089.6 or gp12089.6, with CD4 and CCR5 or CXCR4. We found that gp140(gp120)-CD4-CCR5 complexes are stable and immunogenic in mice transgenic for human CD4 and CCR5. They elicited anti-gp120 and anti-gp140 antibodies that inhibited an heterologous primary HIV-1 isolate (JR-FL) with two- to threefold higher neutralizing activity than those elicited by gp120 and gp140. The antibodies elicited by the complexes competed better with the antibodies X5 and CG10 but not with b12 for binding to gp120 and gp120-CD4 complexes compared to those elicited with gp140(120) alone. These findings suggest that stable purified Env-CD4-CCR5(CXCR4) complexes can be produced in relatively large amount sufficient for their further characterization that may help in the development of novel vaccines candidates. [Copyright &y& Elsevier]

Shows that the detergent-solubilized, purified chemokine receptor CCR5 can directly associate with purified soluble fragments of the extracellular portion of the T cell co-receptor CD4. Result of the physical association of CCR5 and CD4 in membrane vesicles as the formation of a receptor complex that exhibits macrophage inflammatory protein 1β binding properties that are distinct from CCR5.

The first evidence in humans that a safe and effective preventive vaccine for HIV is possible came from the phase III HIV clinical trial RV144 in Thailand. This trial was based on a prime/boost combination of a recombinant canarypox vaccine and two glycoprotein 120 proteins (ALVAC-HIV and AIDSVAX B/E). A pivotal phase IIb/III trial has recently commenced in the Republic of South Africa, for which the infectious titer assay was applied as the quantitative release test for the ALVAC-HIV vaccine. The infectious titer assay measures the ability of the vaccine vector to infect target permissive cells, but does not indicate if the vaccine transgenes are expressed. We have developed a high-throughput biological activity assay that provides results in agreement with the infectious titer assay. This assay uses flow cytometry to quantify expression of ALVAC-HIV encoded proteins gp120 and p24 in human cells. This transgene expression is detected by two cross-clade-reactive, biologically functional human anti-gp120 monoclonal antibodies isolated from clinical trial participants and a commercial mouse anti-p24 monoclonal antibody. The relative biological activity of the vaccine test sample is calculated by comparison of the test sample dose-response curve against that of a reference standard. We show that the novel biological activity assay is specific, accurate, precise, stability-indicating, and robust. The assay is being used for characterization of ALVAC-HIV (vCP2438) product, the efficacy of which is being evaluated in the pivotal phase IIb/III clinical trial HVTN702. The biological activity assay has the potential to indicate vaccine consistency and quality as a complement to the infectious titer assay. [ABSTRACT FROM AUTHOR]

Carbohydrates are abundantly expressed on the surface of both eukaryotic and prokaryotic cells, often as post translational modifications of proteins. Glycoproteins are recognized by the immune system and can trigger both innate and humoral responses. This feature has been harnessed to generate vaccines against polysaccharide-encapsulated bacteria such as Streptococcus pneumoniae , Hemophilus influenzae type b and Neisseria meningitidis. In cancer, glycosylation plays a pivotal role in malignancy development and progression. Since glycans are specifically expressed on the surface of tumor cells, they have been targeted for the discovery of anticancer preventive and therapeutic treatments, such as vaccines and monoclonal antibodies. Despite the various efforts made over the last years, resulting in a series of clinical studies, attempts of vaccination with carbohydrate-based candidates have proven unsuccessful, primarily due to the immune tolerance often associated with these glycans. New strategies are thus deployed to enhance carbohydrate-based cancer vaccines. Moreover, lessons learned from glycan immunobiology paved the way to the development of new monoclonal antibodies specifically designed to recognize cancer-bound carbohydrates and induce tumor cell killing. Herein we provide an overview of the immunological principles behind the immune response towards glycans and glycoconjugates and the approaches exploited at both preclinical and clinical level to target cancer-associated glycans for the development of vaccines and therapeutic monoclonal antibodies. We also discuss gaps and opportunities to successfully advance glycan-directed cancer therapies, which could provide patients with innovative and effective treatments. [ABSTRACT FROM AUTHOR]

b12, one of the few broadly neutralizing antibodies against HIV-1, binds to the CD4 binding site (CD4bs) on the gp120 subunit of HIV-1 Env. Two small fragments of HIV-1 gp120, b121a and b122a, which display about 70% of the b12 epitope and include solubility-enhancing mutations, were designed. Bacterially expressed b121a/b122a were partially folded and could bind b12 but not the CD4bs-directed non-neutralizing antibody b6. Sera from rabbits primed with b121a or b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity in a TZM-bl assay against a 16-virus panel that included nine Tier 2 and 3 viruses as well as in a five-virus panel previously designed to screen for broad neutralization. Using a mean IC50 cut-off of 50, sera from control rabbits immunized with gp120 alone neutralized only one virus of the 14 non-Tier 1 viruses tested (7%), whereas sera from b121a- and b122a-immunized rabbits neutralized seven (50%) and twelve (86%) viruses, respectively. Serum depletion studies confirmed that neutralization was gp120-directed and that sera from animals immunized with gp120 contained lower amounts of CD4bs-directed antibodies than corresponding sera from animals immunized with b121a/b122a. Competition binding assays with b12 also showed that b121a/2a sera contained significantly higher amounts of antibodies directed toward the CD4 binding site than the gp120 sera. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals. [ABSTRACT FROM AUTHOR]