This article discusses the results of neutralization experiments conducted to test human antiV3 monoclonal antibody (mAb) against pesudeoviruses which expressed Env protein of different subtypes.
Catarina E. Hioe1,2*, Terri Wrin3, Michael S. Seaman4, Xuesong Yu5, Blake Wood5, Steve Self5, Constance Williams1, Miroslaw K. Gorny1, Susan Zolla-Pazner1,2
1 Department of Pathology, New York University Langone School of Medicine, New York, New York, United States of America, 2 Veterans Affairs New York Harbor
Healthcare System, New York, New York, United States of America, 3 Monogram Biosciences, Inc., South San Francisco, California, United States of America, 4 Department of Medicine, Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America, 5 Public Health Sciences Division, Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America
Before starting this article, let me quickly revise the structure of HIV virus (Figure). HIV consists of an outer envelope, from which 72 spikes which are formed of gp 120 and gp 41 are projected. Just below the envelope is present matrix, which is made up of the protein p17. The capsid of the virus is bullet shaped and made up of p24. With in the capsid are present enzymes and two identical strands of RNA.
Figure (Source: http://www.avert.org/aids-picture.php?photo_id=504)
Gp120 has been shown to be a critical target for neutralizing antibodies. It serves a crucial function in the pathogenesis of the virus, i.e. as an attachment protein for attachment of the virus to the host cell. Because gp120 plays such important role in virus pathology, it is logical to think that it as a potential target for neutralizing antibodies. However, gp120 displays high antigenic and genetic variability while its conserved regions are poorly immunogenic and are not assessable on the surface of virion at all the time. The polypeptide sequence of this virus consists of five highly variable regions (V1to V5) which are interspersed with five relatively conserved regions. The first four variable regions form disulphide bonded loops which are thought to be flexible.
In the present study, the authors have evaluated the extent and potency of virus neutralization by mAbs specific for epitopes in the V3 loop. The V3 loop was identified as the target of neutralizing antibodies but it was considered inappropriate as it shows high sequence variability. Its importance as a principle neutralizing domain is controversial. Given the fact that V3 is a part of gp120 that interacts with chemokine receptors, it must have some conserved structural elements and it must be exposed, at least transiently, for binding of the virus to the receptors. These features might explain the results of some studies where, many anti-V3 mAbs were able to recognize and neutralize diverse HIV-1 isolates.
Thus, the author tested seven anti-V3 mAbs that have been previously shown to have potent and cross clade neutralizing activity. They carried out neutralization experiments in two independent labs. For this, 98 pseudo viruses (psVs) expressing Envs of HIV-1 subtypes A. AG, B, C, and D from patients with acute and chronic HIV-1 infections were tested.
Positive neutralization was determined by each mAb/psV pair on the basis on commonly used criteria, the 50% mAb inhibitory concentration Ic50. In addition, a new statistical based criteria was used which was based on
1. the area under the mAb titration curve (AUC)
2. the slope of the titration curve
3. the background neutralization from irrelevant control mAbs
4. the background from a control psV expressing Env from amphotropic murine leukaemia virus (aMLV)
Methods:
Seven human anti-V3 mAbs were selected from more than 50 mAbs generated in the labs of the authors. Human monoclonal antibodies specific for parvovirus B19 or anthrax protective antigen (PA) were used as negative control.
The HIV neutralization assays
Neutralization Assays with U87 target cells
These were performed using U87 target cell lines expressing CD4, CCR5 and CXCR4. This assay was used to test 57 psVs expressing cloned Env gene populations extracted from viruses in patients plasma. For the neutralization assay, the psVs were first treated with 2 or 3 fold serial dilutions of mAbs and then incubated with the U87 cells. After 72 hrs of infection, the levels of the virus were assessed by measuring the luciferase activity.
Negative controls: anti parvo virus mAb and aMLV Env-expressing psV.
Positive controls: psVs expressing cloned Envs of SF162, JR-CSF and NL4.3.
Neutralization Assays with TZM.bl target cells
These were performed using TZM.bl target cell lines expressing CD4, CCR5 and CXCR4.
Neutralizing activities of the anti-V3 mAb against 41 psVs containing cloned Env genes from neutralization sensitive viruses (tier 1), from clade B and clade C isolates from recent infections (tier 2) and viruses from chronic infections (tier 3) were measured.
The procedures were similar to those for neutralization assays with U87 target cells.
Negative controls: MAbs specific for parvovirus B19 or anthrax protective antigen were tested.
Results
Comparison of AUC and IC50 values for identifying virus neutralizing activities of anti-V3 mAbs in the U87 assay: When the AUC based statistical analysis was performed, it resulted in the detection of nearly twice as much neutralizing activity as did IC50.
Neutralizing activities of anti-V3 mAbs against multiple HIV-1 subtypes are detected in U87 assay: Of the 57 psVs tested, 21 of the 27 (78%) psVs with subtype B Envs from acute and chronic infection and 8 of the 10 psVs (80%) with subtype C Envs were neutralized by at least one anti-V3 mAb. While, only one out of the 10 subtype D (10%) and 3 of the 10 subtype A (30%) psVs were neutralized by these.
Neutralizing activities of anti-V3 mAbs against multiple HIV-1 subtypes are also detected in TZM.bl assay: When same mAbs were tested against remaining 41 psVs, which included 10 tier1, 12 tier 2 subtype B, 12 tier 2 subtype C and 7 tier 3, it was observed that higher levels of neutralization were detected in the TZM.bl assay than in U87 assay. When 50% neutralization was used as a cut off, 22 of 41 psVs (54%) were neutralized.
Combined data from U87 and TZM.bl assays demonstrate broad neutralizing activities of antiV3 mAbs across Envs from different subtypes and from different stages of infection: When the investigators applied AUC statistical approach to the two independent experiments, 56 0f 98 (57%) psVs were found to be neutralized significantly by more than one anti-V3 mAbs. Now what were these 56 psVs? It was found that these 56 pSVs expressed Envs of diverse subtypes including subtypes A, AG, B, C, and D. thus, the ability of many anti-V3 mAbs to neutralize different subtypes of HIV-1 was observed in both the experiments.
Different antiV3 mAbs display unique patterns of neutralization: It has been seen in previous studies that anti-V3 mAbs derived from African donor infected with HIV-1 non-B subtypes showed different patterns of psV neutralization than those obtained from subtype B infected individuals. It has also been noted that antibodies derived from non-B subtype infected subjects showed broader and more potent neutralization against non-B viruses. In the present study, four non-B derived mAbs were studied. Out of these, two mAbs showed unique neutralization patterns. They showed remarkable neutralization against subtype C psVs. In contrast, they showed sporadic and less potent neutralization against acute, chronic and tier 2 subtype B viruses.
Conclusions:
In the present study, the authors showed that six of the seven anti-V3 mAbs displayed cross clade neutralizing activities. They also observed that 57% of the pseudo viruses tested in this study were effectively neutralized by these mAbs. The authors used old as well as new statistical methods of analysis and observed that this new method has many advantages over the old ones.
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