Maria P. Carlos

Morphine Induces Gene Expression of CCR5 in Human CEM x174 Lymphocytes

All HIV-1 strains studied to date use CCR5, CXCR4, or both receptors to enter cells. Simian immunodeficiency virus (SIV) infection of non-human primates has served as a useful model for understanding AIDS pathogenesis in humans. Research on several genetically divergent SIV isolates has revealed that SIV uses CCR5, and not CXCR4, for entry. CEM x174, a human lymphoid cell line, has been routinely used to cultivate and maintain various SIV strains. However, questions have arisen about how CEM x174, which reportedly was unable to express detectable amounts of CCR5 transcripts, efficiently supports the growth of SIV. In searching for an answer, we resorted to a sensitive competitive reverse transcriptase-polymerase chain reaction procedure in an attempt to detect as well as quantify the amount of CCR5 expression. Here we present our findings, which indicate that CEM x174 indeed expresses CCR5 and that the amount of CCR5 is increased in cells pretreated with morphine. These results correlate well with our previous observations that morphine treatment causes CEM x174 cells to be more susceptible to SIV infection. Similar morphine effect was not observed on CEM x174 cells infected with simian retroviruses, which do not depend on CCR5 for entry. These findings suggest a plausible mechanism whereby opiate drug users render themselves more susceptible to HIV infection, thereby explaining the vast prevalence of HIV infection among endemic drug use populations.

Methadone induces CCR5 and promotes AIDS virus infection

Methadone, a regimen for the treatment of opioid dependency, was found to induce the expression of CCR5, a co‐receptor for human immunodeficiency virus (HIV)/simian form of HIV (SIV) entry, on human CEM x174 lymphocytes. Both CCR5 mRNA and protein were elevated in methadone‐treated cells. A concomitant increase of mu opioid receptors was also observed. Upon methadone exposure, SIVmac239‐infected CEM x174 cells released greater amounts of virus particles as revealed by both the number of syncytia formation and reverse transcriptase activities. Similar methadone effect was not observed on CEM x174 cells infected with other simian retroviruses that do not depend on CCR5 for cellular entry. These studies raise concerns considering methadone as an innocuous morphine substitute.

Immunogenicity of a vaccine preparation representing the variable regions of the HIV type 1 envelope glycoprotein.

Variability of the major antigenic sites of the envelope glycoprotein of HIV-1 constitutes a major problem in the formulation of effective vaccines. We have prepared a synthetic peptide vaccine that represents the major hypervariable epitopes (V1 through V5) of the clade B HIV-1 envelope glycoprotein (gp120). We refer to this preparation as variable epitope immunogen or VEI vaccine. This construct takes into consideration the type and frequency of amino acid substitutions found at each epitope during the evolution of the virus in individual patients and in the target population. Immunization of mice, rabbits, and rhesus macaques with the VEI vaccine resulted in the induction of long-lasting, high-titered HIV-1 antibodies, including antibodies that neutralize primary isolates. We also documented lymphocyte proliferative responses to the VEI vaccine, its individual components, analogs, and subtype-specific peptides representing the major hypervariable regions of HIV-1 gp120. Delayed-type hypersensitivity responses to these antigens were also demonstrated in mice. Our results show that this vaccine is highly immunogenic and safe in animals. Our data suggest that this formulation could become an important component of combination vaccine approaches against HIV-1 and other antigenically variable pathogens.

Effect of Human Immunodeficiency Virus Type 1 on Intracellular Activation and Superoxide Production by Neutrophils

The immunopathogenesis of AIDS is associated with the development of opportunistic infections by intracellular pathogens that can invade and reproduce freely because of impaired cellular functions. Neutrophils from asymptomatic human immunodeficiency virus (HIV) type 1-infected persons and from symptomatic patients with AIDS were found to retain normal phagocytosis activity while producing significantly less superoxide than neutrophils from HIV-1-negative subjects, when stimulated through Fc receptors or protein kinase C. After priming with a synthetic HIV-1 envelope peptide and stimulation via the Fc receptor, the neutrophils from HIV-1-negative controls had suppressed superoxide production, reduced phosphorylation of two unidentified cellular proteins, and increased expression of a third phosphoprotein. These results suggest that HIV-1 can produce direct functional damage of neutrophils through binding of envelope components to the cell membrane.

Immunological and chemical analysis of proteins from Eisenia foetida earthworm

Previous studies have demonstrated the fibrinolitic and antibacterial activities of proteins from the earthworm Eisenia foetida (Ef) but their use as a food supplement for animals and humans is still being considered. We studied proteins from this worm in order to determine chemical composition (protein, fat and heavy metal levels), electrophoretic profile in SDS-PAGE and 2D gels, induction of humoral immune response in mice and toxicity in a human cell line. Chemical analysis revealed 61.8, 11.3 and 8.7 g% of proteins, fat and ash, respectively. Levels of heavy metals were low and similar to tunny fish. Sixteen bands were obtained by SDS-PAGE with a molecular weigh range of 6.9–205 kDa while twenty-three spots were observed following 2D gel electrophoresis. After three immunizations with Eisenia foetida lysate, high titer antibodies were produced without causing immediate hypersensitivity reactions. Good cell viability was observed when Ef proteins were added to a human cell line. These results suggest that Ef proteins are safe for feeding animals intended for human consumption.

Antibodies from HIV-positive and AIDS patients bind to an HIV envelope multivalent vaccine.

A major problem impeding development of an effective HIV vaccine is the rapid antigenic variability that is characteristic of several envelope glycoprotein epitopes. Frequent mutations alter the composition of the most immunogenic regions of the envelope glycoprotein. We have prepared a synthetic immunogen representing the evolution of the major hypervariable epitopes on the envelope glycoprotein (gp120) of HIV-1. Five synthetic constructs, representing each of the HIV-1 gp120 hypervariable epitopes were tested for recognition by antibodies from patients infected with HIV-1 from different geographic regions worldwide. An HIV-1 human plasma panel provided a representation of the antibodies recognizing subtype-specific epitope sequences prevalent at different parts of the world. The vaccine construct was recognized by antibodies from HIV-1-positive individuals infected with subtypes A, B, C, D, E, and F. Antibodies in pooled HIV-1 patient sera from San Francisco also recognized all five constructs. This complex immunogen was recognized by antibodies in sera from individual HIV-1-positive and AIDS patients from Puerto Rico and Canada, with a strong binding to the complete vaccine and the V3 component. Altogether, our results demonstrate that antibodies from seropositive patients infected with different HIV-1 clades recognize and bind to the HIV hypervariable epitope construct vaccine preparation and its individual components.

Induction of immune responses against human papillomaviruses by hypervariable epitope constructs

An ideal prophylactic vaccine against human papillomaviruses (HPV) would be one that can induce broadly reactive antibody titres to at least the major oncogenic strains of HPV. It has been previously shown that HPV structural proteins are highly immunogenic but fail to elicit cross‐reactive immune responses against heterologous strains of HPV. Recent studies have demonstrated that the immunity induced by virus‐like particles is mostly type specific. In the present study, we determined the breadth of reactivity of antibodies induced in mice immunized with hypervariable epitope constructs (HECs), which represent sequence variants of immunodominant B‐cell epitopes of the major capsid protein L1 of HPV. In order to test the breadth of reactivity, sera from immunized mice were tested against peptides representing analogous sequences of HPV types 16, 18, 31 and 45. Mice immunized with HECs based on two epitopes mounted antibody responses that cross‐reacted with two different analogues, 16 and 18. Significantly, antibodies from mice immunized with HECs also inhibited haemagglutination mediated by HPV‐16 L1 VLPs, suggesting that immunization resulted in the development of antibodies that could bind to viral capsid proteins in their native conformation. Our observations suggest that HECs may overcome the restriction of type specific immunity against HPV.

Synthetic Antigens Representing the Antigenic Variation of Human Hepatitis C Virus

Immune responses against hepatitis C virus (HCV) have been studied by numerous groups. However, details concerning the production of antibodies to antigenically variable epitopes remain to be elucidated. Since the sequences of the variable regions of several HCV proteins are different among the virus strains infecting patients, we decided to design peptide combinations that represent the theoretical maximum antigenic variation of each epitope to be used as capture antigens. We prepared six peptide mixtures (hypervariable epitope constructs; HECs) representing six different epitopes from structural and non-structural proteins of HCV from genotypes 1-6. Plasma from 300 HCV patients was tested to determine if their antibodies recognize the synthetic constructs. All the patients were chronically infected with diverse HCV genotypes and did not receive antiviral treatment. Antibodies to one or more of the HECs were detected in all of the HCV-infected individuals. Immunogenicity of the HCV HECs was also evaluated in outbred and inbred mice. Strong HEC-specific antibodies were produced, and cellular responses were also induced that were Th-1 rather than Th-2. Our results show that HCV HECs are both antigens that can be used to detect the broad cross-reactivity of antibodies from HCV-infected patients, and strong immunogens that can induce antigen-specific humoral and cellular immune responses in mice.