REPORT FOR THE PFIZER FOUNDATION

  • Name: MELISSA SOARES MEDEIROS
  • Dates of training fellowship at UVa: March 2006 - February 2007.
  • Home Institution: Federal University of Ceara, Fortaleza, Ceara, Brazil.
  • Title/position there before coming to UVa: Physician with residence in Infectious Diseases (February/2006), master degree in pharmacology (February/2006) and PHD in pharmacology (in course, started in February/2006).
  • Paragraph summary of training at UVa

Dr. Medeiros's work was conducted at the Myles H. Thaler Center for AIDS and Human Retroviruses, under the mentorship of Drs. David Rekosh and Marie-Louise Hammarskjöld. They focused on three projects:

  1. Generating data on baseline resistance of HIV from Haiti children with drug resistance to currently used protease and reverse transcriptase inhibitors - Plasma samples were collected from 13 children from Haiti suspected to be resistant to antiretroviral drugs.
  2. Viral Infection of Sup T1 Cells with T20 and 104833 (Rev inhibitor drug) - in vivo resistance effect in HIV virus 1272 and 1310.
  3. Characterize and evaluate the constraints imposed on fusion inhibitor resistance and relate it to the RRE/Rev.

    Research activities and outcomes during training

1) Generating data on baseline resistance of HIV from Haiti children with drug resistance to currently used protease and reverse transcriptase inhibitors - Plasma samples were collected from 13 children from Haiti suspected to be resistant to antiretroviral drugs.

Methodology: The complete protease (PR) gene and reverse transcriptase (RT) gene of 13 viruses were amplified by reverse transcriptase PCR, sequenced, analyzed with Stanford Data Base and examined for mutations associated with resistance.

In our final report three samples could not be amplified, we supposed they had very low viral load (probable undetectable).

Results: After analysis of sequences from PR and RT patients, we found resistance to drugs as reported below:

Resistance to NRTI: Lamivudine (30% High resistance, 10% intermediary resistance and 60% susceptible), Abacavir (20% High resistance, 30% low resistance and 50% susceptible), Zidovudine (20% High resistance, 10% low resistance and 70% susceptible), Stavudine (20% High resistance, 20% low resistance and 60% susceptible), Didanosine (10% High resistance, 20% intermediary resistance, 10% low resistance and 60% susceptible), Emtricitabine (30% High resistance, 10% intermediary resistance and 60% susceptible), Tenofovir (30% Intermediary resistance, 10% low resistance and 60% susceptible).

Resistance to this class was attributed to NAM (nucleoside analogous mutations) accumulation (41L, 67N, 70R, 210W, 215Y/F, 219E/Q, 44D and 118I). We found K65R in just one patient, so resistance to Tenofovir occurred for NAM accumulation.

Resistance to NNRTI: Efavirenz (10% High resistance, 50% intermediary resistance and 40% susceptible), Nevirapine (10% High resistance, 50% intermediary resistance, 10% low resistance and 30% susceptible).

The most common resistance mutation to this class is K103N which was found in just one patient, so resistance was attributed to other resistances to this class (101I,106M, 181C/I and 188C/L/H).

Resistance to Protease Inhibitors: 100 Susceptible to Atazanavir, Indinavir, Lopinavir, Nelfinavir, Ritonavir, Saquinavir, Tipranavir and for Amprenavir. L63P was found in just two sequences. So, this is a polymorphic mutation which is more common in experienced patients. We did not have the patients' previous drug history. We supposed they probably did not use PI before or had suspended use before we collected the plasma sample.

Conclusion: We concluded these patients did not have very high resistance to antiretroviral drugs, perhaps because they could not take them properly before collecting the samples. And we know drug selection pressure is important to resistance evaluation. Also, the higher resistance to the NRTI class could be attributed to previous use of these in therapeutic treatments and not in use of PI class.

2) Viral Infection of Sup T1 Cells with T20 and 104833 (Rev inhibitor drug) - in vivo resistance effect in HIV viruses 1272 and 1310.

Methodology: Sup T1 cells were infected with viral stocks of the two different viruses (1272 and 1310) and infected cultures were maintained in three different concentrations of drug. The first concentration approximated the IC-50 of T20, the next the IC-90 and the highest concentration used was three times the IC-50 concentration:

- T20 1 μg/20ml of 0,2 μg/ml of work solution

- T20 10 μg/20ml of 0,2 μg/ml of work solution

- T20 30 μg/20ml of 0,2 μg/ml of work solution

- 104366 5ml 8μm

T-20 is a fairly new class compound introduced in HIV therapeutic, which targets the gp41 membrane protein, playing a critical role in HIV fusion to the host cell.  104366 is a new compound in study at UVa's Myles T. Center described as a possible Rev gen inhibitor.

A control was also performed where cultures were maintained in the absence of drugs. Infected cells were maintained in culture by removal of the media every two to three days, with the addition of the same volume of fresh media. To follow the infection, p24 ELISA assays were performed at three day intervals.

Results: We found in the absence of drugs, peak levels of p24 occurred around day seven for both viruses. In the presence of drugs, viral replication was delayed in all cases. For the two highest concentrations of drugs, viral suppression was significant and lasted for several days. However, virus replication was eventually observed in most cultures. These viruses are presently being analyzed for resistance to T-20. The re was not growth virus with 104 drug in both viruses used.

Conclusion: Our preliminary results show both viruses can be suppressed in Sup T1 cells, using a T-20 fusion inhibitor. The increase in viral replication after many days of suppression indicates that resistant variants might have been generated.

3) Characterize and evaluate the constraints imposed on fusion inhibitor resistance and relate it to the RRE/Rev.

This is a new project that has been developed in Brazil with UVa support.

The long-range goal of this study is to understand how gp41 mutates to resistance in response to inhibitors that target viral fusion and to study the requirement for maintenance of RRE function in the mutated viruses. The work may have long-term clinical importance as it will address the validity of the concept of combination therapy, using a fusion inhibitor and a Rev inhibitor and the potential for synergy in such protocols.

Material and Methods:  Study population: HIV/AIDS patients being treated with T20 in rescue antiretroviral regimens.

Local: Charlottesville clinics (USA) and Hospital Sao Jose in Fortaleza (Brazil).

Ethics Committee: The protocol was submitted to Ethics committees in the United States and Brazil. The Brazilian committee has extended its approval.

Protocol:

*Identify patients taking T20 in Antiretroviral Therapy

*Review Files with epidemiologic, clinical and laboratorial data

*Take a blood sample for genotyping prior to initiating therapy with T20

*Follow up with patients each month, when they come back to the hospital to get new T20 receipts, evaluate therapy adherence and side effects. Take a new blood sample every three to four months to get viral load or if patients present signs of clinical failure.

*Once resistance is suspected, another blood sample should be drawn for genotyping. 

The effect of resistance on HIV fitness would then be studied in the Hammarskjold-Rekosh lab at UVa using standard fitness assays. Additionally, the lab has methods to measure "RRE fitness" specifically, including a method of producing viruses in which the RRE is uncoupled from the gp41 sequence. By examining the effect of mutations conferring resistance to T20 in these assays, it should be possible to deduce if maintaining a functional RRE is a constraint on the generation of T20 resistance.

  • Meetings, conferences, workshops, courses attended.

I gave a seminar at UVa's Center for Global Health entitled Genotyping test and Antiretroviral profile of Human Immunodeficiency Virus type 1 isolated from failure therapy patients in Brazil - 2002 to 2004 (May, 2006).

  • Presentations

American Society of Tropical Medicine and Hygiene. 55th Annual Meeting. Nov 15, 2006.  Poster 826: IMPACT OF FAILED THERAPEUTIC REGIMES IN THE RESISTANCE TO ANTIRETROVIRAL DRUGS IN NORTHEST BRAZIL

Melissa Soares Medeiros1,2,3, Érico Antônio Gomes Arruda2, Richard Guerrant3 and Aldo Ângelo Moreira Lima1,3

Federal University of Ceara1; São José Hospital of Infectious Diseases2; Fortaleza, CE, Brazil; University of Virginia-Center for Global Health, Charlottesville VA, USA3

  • Publications and papers in preparation:

1. GENOTYPIC TEST AND ANTIRETROVIRAL RESISTANCE PROFILES FROM HIV- 1 PATIENTS EXPERIENCING THERAPEUTIC FAILURE IN NORTHEAST BRAZIL. Melissa Soares Medeiros1,2, 3, Christopher Brown 3, Érico Antônio Gomes Arruda2 , Richard Littleton Guerrant3, Aldo Ângelo Moreira Lima1, 3

Universidade Federal do Ceará, Departamento de Farmacologia e Fisiologia, Fortaleza, IBIMED, CE, Brazil1; Hospital São José de Doenças Infecciosas-SESA, Fortaleza, CE, Brazil 2; University of Virginia, Center of Global Health, Charlottesville, VA, USA3. (In preparation for Brazilian Journal of infectious Diseases).

2. IMPACT OF FAILED THERAPEUTIC REGIMES ACCUMULATION IN THE DEVELOPMENT OF RESISTANCE MUTATIONS TO HIV-1 IN NORTHEST BRAZIL. Melissa Soares Medeiros1,2, 3, Christopher Cooley Brown3, Érico Antônio Gomes Arruda2 , Richard Littleton Guerrant3, Aldo Ângelo Moreira Lima1, 3

Universidade Federal do Ceara, Departamento de Farmacologia e Fisiologia, Fortaleza, IBIMED, CE, Brazil1; Hospital São José de Doenças Infecciosas2; Fortaleza, CE, Brazil; University of Virginia, Center of Global Health, Charlottesville, VA, USA3. (In preparation for Brazilian Journal of infectious Diseases).

  • Future plans - personal plans, research plans, and institutional collaboration with UVa

During my master degree work, I studied genotyping tests that were performed in HIV patients failing therapy. As I had worked mainly in clinical research, I decided to turn my attention to basic and molecular research. So I decided to come to UVa to improve my skills so i would be better suited to return home and improve my research center in Brazil.

My personal plans are to return to my PHD program in Brazil and develop new projects based on many protocols and techniques I had been trained in at UVa, like viruse cultures and Genotyping assays. Happily, we have a sequence machine in the Brazilian center that enable us to develop new resistance protocols in our country. Because we work together with the reference HIV/AIDS hospital we will be able to study resistance in native patients, pregnant women and newborns admitted to antiretroviral therapy as well as patients taking a new class of drugs.

My plans also include helping to adapte a laboratory in Brazil so I and my collegues can work with viral cultures of HIV. We will be assisted in this by my UVa mentors, Drs. Rekosh and Hammarskjöld.

  • Mentoring - describe how you worked with other fellows in the lab, and/or other fellows or UVa students.

Built on strong research and field programs with increasingly active collaborative research and exchanges since 1978, our UFC/UVA Program has developed into a true collaboration that has formed the basis of lasting institutional strengths at both the Federal University of Ceara (UFC) and the University of Virginia (UVA). Over the years, a steady progression of faculty, fellow and student exchanges between our Universities and increasingly productive collaborations with special focus on enteric, parasitic, respiratory and nosocomial infections have led to the development of established programs at each of our institutions. 

During my Pfizer Fellowship work at UVa, I've had the opportunity to conduct lba work with researchers from the United States, as well as from Africa. I felt very accepted in from the moment I arrived in Charlottesville. I received personal support to from my mentors and also from the Center of Global Health. During this time we built strong bridges among ours students that will help us to keep this collaborative program in the future.

Now that I am back in Brazil, it is my goal to continue to collaborate with these scientists on research that will both make important new discoveries in the treatment of HIV/AIDS and create a long-lasting alliance between our two Universities.