M Patricia D'Souza

Publications of M Patricia D'Souza

• Development and implementation of an international proficiency testing program for a neutralizing antibody assay for HIV-1 in TZM-bl cells.

  Authors: Christopher A Todd, Kelli M Greene, Xuesong Yu, Daniel A Ozaki, Hongmei Gao, Yunda Huang, Maggie Wang, Gary Li, Ronald Brown, Blake Wood, M Patricia D'Souza, Peter Gilbert, David C Montefiori, Marcella Sarzotti-Kelsoe

  Journal of immunological methods. 01/2012; 375(1-2):57-67.

  Recent advances in assay technology have led to major improvements in how HIV-1 neutralizing antibodies are measured. A luciferase reporter gene assay performed in TZM-bl (JC53bl-13) cells has beenRecent advances in assay technology have led to major improvements in how HIV-1 neutralizing antibodies are measured. A luciferase reporter gene assay performed in TZM-bl (JC53bl-13) cells has been optimized and validated. Because this assay has been adopted by multiple laboratories worldwide, an external proficiency testing program was developed to ensure data equivalency across laboratories performing this neutralizing antibody assay for HIV/AIDS vaccine clinical trials. The program was optimized by conducting three independent rounds of testing, with an increased level of stringency from the first to third round. Results from the participating domestic and international laboratories improved each round as factors that contributed to inter-assay variability were identified and minimized. Key contributors to increased agreement were experience among laboratories and standardization of reagents. A statistical qualification rule was developed using a simulation procedure based on the three optimization rounds of testing, where a laboratory qualifies if at least 25 of the 30 ID50 values lie within the acceptance ranges. This ensures no more than a 20% risk that a participating laboratory fails to qualify when it should, as defined by the simulation procedure. Five experienced reference laboratories were identified and tested a series of standardized reagents to derive the acceptance ranges for pass-fail criteria. This Standardized Proficiency Testing Program is the first available for the evaluation and documentation of assay equivalency for laboratories performing HIV-1 neutralizing antibody assays and may provide guidance for the development of future proficiency testing programs for other assay platforms.

• Quality assurance of intracellular cytokine staining assays: analysis of multiple rounds of proficiency testing.

  Authors: Maria C Jaimes, Holden T Maecker, Ming Yan, Vernon C Maino, Mary Beth Hanley, Angela Greer, Janice M Darden, M Patricia D'Souza

  Journal of immunological methods. 01/2011; 363(2):143-57.

  When evaluating candidate prophylactic HIV and cancer vaccines, intracellular cytokine staining (ICS) assays that measure the frequency and magnitude of antigen-specific T-cell subsets are one toolWhen evaluating candidate prophylactic HIV and cancer vaccines, intracellular cytokine staining (ICS) assays that measure the frequency and magnitude of antigen-specific T-cell subsets are one tool to monitor immunogen performance and make product advancement decisions. To assess the inter-laboratory assay variation among multiple laboratories testing vaccine candidates, the NIH/NIAID/DAIDS in collaboration with BD Biosciences implemented an ICS Quality Assurance Program (QAP). Seven rounds of testing have been conducted in which 16 laboratories worldwide participated. In each round, IFN-γ, IL-2 and/or TNF-α responses in CD4+ and CD8+ T-cells to CEF or CMV pp65 peptide mixes were tested using cryopreserved peripheral blood mononuclear cells (PBMC) from CMV seropositive donors. We found that for responses measured above 0.2%, inter-laboratory %CVs were, on average, 35%. No differences in inter-laboratory variation were observed if a 4-color antibody cocktail or a 7-color combination was used. Moreover, the data allowed identification of important sources of variability for flow cytometry-based assays, including: number of collected events, gating strategy and instrument setup and performance. As a consequence, in this multi-site study we were able to define pass and fail criteria for ICS assays, which will be adopted in the subsequent rounds of testing and could be easily extrapolated to QAP for other flow cytometry-based assays.

• Acute HIV-1 infection: what's new? Where are we going?

  Authors: M Patricia D'Souza, Karen L Axten, Frederick M Hecht, Marcus Altfeld

  The Journal of infectious diseases. 10/2010; 202 Suppl 2:S267-9.

  This supplemental issue of the Journal of Infectious Diseases is devoted to the important topic of primary human immunodeficiency virus type 1 (HIV-1) infection. It was prompted by the planning ofThis supplemental issue of the Journal of Infectious Diseases is devoted to the important topic of primary human immunodeficiency virus type 1 (HIV-1) infection. It was prompted by the planning of the Acute HIV-1 Infection Meeting in Boston in September 2009, at which leading scientists and practitioners gathered to discuss new insights into the early, critical events of HIV-1 infection. The reviews that follow underline the current state of the field with regard to transmission biology of HIV-1; the clinical presentation, diagnosis, and management of primary HIV-1 infection; the pathogenesis of primary HIV-1 infection; and innate and adaptive immune responses to the virus. We trust that these findings have the potential to influence the development of effective vaccine strategies.

• Challenges for rapid molecular HIV diagnostics.

  Authors: Marco L Schito, M Patricia D'Souza, S Michele Owen, Michael P Busch

  The Journal of infectious diseases. 04/2010; 201 Suppl 1:S1-6.

  The introduction of serological point-of-care assays 10 years ago dramatically changed the way that human immunodeficiency virus (HIV) infection was identified and diagnosed. Testing at the point ofThe introduction of serological point-of-care assays 10 years ago dramatically changed the way that human immunodeficiency virus (HIV) infection was identified and diagnosed. Testing at the point of care has lead to a dramatic increase in the number of individuals who are screened and, most importantly, receive their HIV test result. As the AIDS epidemic continues to mature and scientific advances in prevention and treatment are evaluated and implemented, there is a need to identify acute (viremic preseroconversion) infections and to discriminate "window phase" infections from those that are serologically positive, especially in resource-limited settings, where the majority of vulnerable populations reside and where the incidence of HIV infection is highest. Rapid testing methods are now at a crossroads. There is opportunity to implement and evaluate the incremental diagnostic usefulness of new test modalities that are based on sophisticated molecular diagnostic technologies and that can be performed in settings where laboratory infrastructure is minimal. The way forward requires sound scientific judgment and an ability to further develop and implement these tests despite a variety of technical, social, and operational hurdles, to declare success.

• Measuring HIV-1-specific T cell immunity: how valid are current assays?

  Authors: M Patricia D'Souza, Marcus Altfeld

  The Journal of infectious diseases. 03/2008; 197(3):337-9.

• Current advances and challenges in HIV-1 vaccines.

  Authors: Isaac R Rodriguez-Chavez, Mary Allen, Edgar L Hill, Rebecca L Sheets, Michael Pensiero, James A Bradac, M Patricia D'Souza

  Current HIV/AIDS reports. 03/2006; 3(1):39-47.

  Recent advances in science, which have aided HIV-1 vaccine development, include an improved understanding of HIV-1 envelope structure and function, expansion of the pipeline with innovative vaccineRecent advances in science, which have aided HIV-1 vaccine development, include an improved understanding of HIV-1 envelope structure and function, expansion of the pipeline with innovative vaccine strategies, promising multi-gene and multi-clade vaccines that elicit cellular immunity, conduct of clinical trials in a global network, and development of validated techniques that enable simultaneous measurement of multiple T cell vaccine-induced immune responses in humans. A common feature of several preventive vaccine strategies now in early clinical trials is their ability in nonhuman primates to attenuate clinical disease rather than completely prevent HIV-1 infection. One vaccine concept has been tested in large-scale clinical trials, two are currently in efficacy trials, and one more is poised to enter efficacy trial in the next few years. Simultaneously, expanded efforts continue to identify new designs that induce mucosal immunity as well as broadly neutralizing antibodies.

• Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials.

  Authors: Josephine H Cox, Guido Ferrari, Spyros A Kalams, Wlodek Lopaczynski, Neal Oden, M Patricia D'souza

  AIDS research and human retroviruses. 02/2005; 21(1):68-81.

  We used an external quality assurance (EQA) panel to assess laboratory competency and comparability when performing ELISPOT assays in support of human immunodeficiency virus type 1 (HIV-1) vaccineWe used an external quality assurance (EQA) panel to assess laboratory competency and comparability when performing ELISPOT assays in support of human immunodeficiency virus type 1 (HIV-1) vaccine trials. Cell recovery, viability, and frequency of interferon-gamma (IFN-gamma)-secreting cells after antigen stimulation were obtained from 11 laboratories on a coded panel of 11 peripheral blood mononuclear cell samples. The median recovery and viability before plating for all samples were 35% and 86%, respectively, with notable interlaboratory and intrasample variability. Empirical as well as statistical analysis methods were used to define positive ELISPOT responses. Remarkable concordance between laboratories was obtained in defining a qualitative assessment of responder/nonresponder status to antigens, but the frequency of responding cells varied among the laboratories. This study highlights the need for better standardization of protocols and reagents to obtain reliable and reproducible data that may support immunogenicity studies, vaccine regulatory submissions, and licensure.

• Current advances in HIV vaccines.

  Authors: M Patricia D'Souza, Mary Allen, Rebecca Sheets, Margaret I Johnston

  Current HIV/AIDS reports. 05/2004; 1(1):18-24.

  Development of a safe and preventive HIV-1 vaccine is a high priority. Recent advances in HIV vaccine development include an improved understanding of HIV envelope structure, development ofDevelopment of a safe and preventive HIV-1 vaccine is a high priority. Recent advances in HIV vaccine development include an improved understanding of HIV envelope structure, development of techniques that enable a detailed analysis of vaccine-induced immune responses in humans, expansion of the pipeline of promising candidate vaccines, and completion of the first vaccine efficacy trials. A common feature of several preventive vaccine strategies in early clinical trials is their ability to attenuate clinical disease rather than completely prevent HIV infection in nonhuman primates. One or more candidate vaccines will likely advance into efficacy trials within the next few years, while efforts to identify new designs that induce broadly neutralizing antibodies continue with incremental success.

• HIV Vaccines: Biological and Clinical Considerations.

  Authors: M Patricia D'Souza, Mary Allen, Margaret Johnston

  Current infectious disease reports. 09/2002; 4(4):359-368.

  The discovery of an HIV-1 vaccine is a high priority. Recent advances in HIV vaccine development include an improved understanding about virus biology and structure, and the development ofThe discovery of an HIV-1 vaccine is a high priority. Recent advances in HIV vaccine development include an improved understanding about virus biology and structure, and the development of quantitative techniques that enable a detailed analysis of vaccine-induced immune responses in humans. The preclinical vaccine pipeline looks healthy, and a common feature of the new vaccine strategies is their ability to attenuate clinical disease rather than prevent HIV infection in nonhuman primates. Human clinical trials to evaluate the safety and immunogenicity of these vaccine candidates and strategies are being actively pursued.