Isolation of the human immunodeficiency virus (HIV) from the cerebro-spinal fluid

The authors describe a simple and available technique for HIV isolation from cerebrospinal fluid and report preliminary results obtained. Exposed data indicate that neurological involvement occurs early in the natural history of HIV infection and that the virus may be recovered in CSF at all stages of the disease, regardless of immunological conditions of patients. Virological evaluation of CSF may be important in understanding pathogenetic aspects of HIV infection and in clinical management of infected patients.     

Removing human immunodeficiency virus (HIV) from human blood using immobilized heparin

Heparin covalently attached to a water-insoluble resin suspended in HIV-infected aqueous buffer or whole blood captures the virus; subsequent physical separation of the immobilized heparin reduced the viral titers by over 80 and 50%, respectively. The detoxification concept has been validated by both circulating an HIV-1 solution through a column packed with the heparin–sepharose beads and successively mixing an HIV-1 solution with fresh beads.     

Isolation of human immunodeficiency virus (HIV) from whole blood]

The authors describe a simple and sensitive technique for HIV isolation from small amounts of heparinized whole blood. This method demonstrated a high efficiency in detecting HIV at all stages of disease and appeared more sensitive with respect to viral isolation from peripheral blood mononuclear cells. Although further studies are needed to better understand the biological significance of a positive cultural result obtained by this method, HIV isolation from whole blood can be routinely employed, especially when small amounts of blood are available.     

The pathogenicity of human immunodeficiency virus (HIV) type 1 Nef in CD4C/HIV transgenic mice is abolished by mutation of its SH3-binding domain, and disease development is delayed in the absence of Hck

The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important determinant of AIDS pathogenesis. We have previously reported that HIV-1 Nef is responsible for the induction of a severe AIDS-like disease in CD4C/HIV transgenic (Tg) mice. To understand the molecular mechanisms of this Nef-induced disease, we generated Tg mice expressing a mutated Nef protein in which the SH3 ligand-binding domain (P(72)XXP(75)XXP(78)) was mutated to A(72)XXA(75)XXQ(78). This mutation completely abolished the pathogenic potential of Nef, although a partial downregulation of the CD4 cell surface expression was still observed in these Tg mice. We also studied whether Hck, one of the effectors previously found to bind to this PXXP motif of Nef, was involved in disease development. Breeding of Tg mice expressing wild-type Nef on an hck(-/-) (knockout) background did not abolish any of the pathological phenotypes. However, the latency of disease development was prolonged. These data indicate that an intact PXXP domain is essential for inducing an AIDS-like disease in CD4C/HIV Tg mice and suggest that interaction of a cellular effector(s) with this domain is required for the induction of this multiorgan disease. Our findings indicate that Hck is an important, but not an essential, effector of Nef and suggest that another factor(s), yet to be identified, may be more critical for disease development.     

Immune responses following simian/human immunodeficiency virus (SHIV) challenge of rhesus macaques after human immunodeficiency virus (HIV)-1 third variable domain (V3) loop-based genetic immunization

Following DNA immunization of rhesus macaques with a plasmid encoding the human immunodeficiency virus (HIV)-1 third variable domain (V3) loop, presented by pseudo-viral envelope particles of hepatitis B virus, specific immune responses were induced. The primates were then inoculated with a chimeric simian/human immunodeficiency virus (SHIV). All the animals were infected, but the V3-specific immunization provided a relative attenuation of the acute phase of infection in the absence of neutralizing antibody. In all animals, SHIV-specific cytotoxic T-lymphocyte precursors (CTLp) were detected early in peripheral blood and lymph nodes. The viremia peak correlated significantly with the decrease in CD4+ T cells and with a transient increase in the percentage of natural killer cells. The infection induced an oligoclonalization of the CD8+ T-cell variable beta chain repertoire in the blood. Surprisingly, HIV envelope-specific CTLp generated by genetic immunization may be governed by distinct circulation rules compared to SHIV-specific CTLp induced by infection.     

Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1.

Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab')(2) fragments or with papain to generate Fab fragments. IgG3 F(ab')(2) fragments were still more efficient in neutralization than F(ab')(2) of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.     

The transmission dynamics of human immunodeficiency virus (HIV)

The paper first reviews data on HIV infections and AIDS disease among homosexual men, heterosexuals, intravenous (IV) drug abusers and children born to infected mothers, in both developed and developing countries. We survey such information as is currently available about the distribution of incubation times that elapse between HIV infection and the appearance of AIDS, about the fraction of those infected with HIV who eventually go on to develop AIDS, about time-dependent patterns of infectiousness and about distributions of rates of acquiring new sexual or needle-sharing partners. With this information, models for the transmission dynamics of HIV are developed, beginning with deliberately oversimplified models and progressing--on the basis of the understanding thus gained--to more complex ones. Where possible, estimates of the model's parameters are derived from the epidemiological data, and predictions are compared with observed trends. We also combine these epidemiological models with demographic considerations to assess the effects that heterosexually-transmitted HIV/AIDS may eventually have on rates of population growth, on age profiles and on associated economic and social indicators, in African and other countries. The degree to which sexual or other habits must change to bring the 'basic reproductive rate', R0, of HIV infections below unity is discussed. We conclude by outlining some research needs, both in the refinement and development of models and in the collection of epidemiological data.     

Impact of epitope permutations in the antibody response of mice to a multi-epitope polypeptide of the V3 loop of human immunodeficiency virus type 1

Our group have produced in Escherichia coli and evaluated the immunogenicity of different multi-epitope polypeptides (MEPs) bearing one copy of V3 loop sequential B cell epitopes from several isolates of human immunodeficiency virus type 1 (HIV-1) gp120. One of these MEPs called TAB9 comprises the 15 central amino acids of the V3 loop from isolates LR150, JY1, RF, MN, BRVA and IIIB in this order. Antibodies against all V3 regions were elicited after immunization of rabbits, macaques and humans with TAB9. In contrast, mice immunized with this protein only developed antibodies against epitopes JY1, LR150 and MN in that order (JY1>LR150>MN>RF, BRVA, IIIB) resembling an immunodominant gradient from the N-terminus to the C-terminal portion of this construction. To assess what role the location of the V3 epitopes in TAB9 could play, we constructed the protein TAB16, by altering the position of V3 epitopes in TAB9 primary structure and compared the pattern of antibodies elicited by both MEPs in H-2(d) Balb/c mice. The MEP TAB16 elicited antibody titers comparable to that of the sera from mice immunized with TAB9. There were no statistical differences in antibody titers between both groups (P>0.05). JY1, LR150 and MN V3 epitopes were again immunodominant in mice immunized with TAB16 fusion protein. The highest antibody titers detected in both groups among V3 epitopes corresponded to JY1, now located at the C-terminus of the permuted chimera. Antibodies against V3 epitopes RF, BRVA and IIIB were again not detected. Additionally, the MN V3 epitope showed to be significantly more immunogenic in its new orientation in TAB16, possibly as a result of a higher degree of accessibility in the surface of the protein. The results of the present investigation strongly suggest that the sequential order or the intramolecular position of V3 epitopes inside the primary structure of TAB9 and TAB16 MEPs does not interfere with the global immunogenicity or with the hierarchy of immunodominance of these regions.     

A kinetic model of CD4+ lymphocytes with the human immunodeficiency virus (HIV).

This report describes a kinetic model of in vitro cytopathology involving interactions of human immunodeficiency virus (HIV) with CD4+ helper T lymphocytes. The model uses nonlinearly coupled, ordinary differential equations to simulate the dynamics of infected and uninfected cells and free virions. It is assumed that resting cells are more readily infected than activated cells, but once infected, only activated cells produce more virus. Resting cells can be activated by some appropriate stimulus (e.g. phytohemagglutinin, soluble antigen). The model predicts that the initial inoculum of virus is taken up by resting cells and without stimulation the system comes to a steady state of two populations, namely infected and uninfected cells. Stimulation of this system produces two additional populations, namely infected and uninfected activated cells which, along with the previous populations, exhibit cyclic behavior of growth, viral expression/release, and death. Additional stimuli enhance or diminish the cyclic behavior depending upon their occurrence in time. These simulations suggest a similar dynamics in human HIV infection and may explain a major factor responsible for the widely varying depletion rate of (CD4+) helper T cells in AIDS patients.     

On the origin and evolution of the human immunodeficiency virus (HIV)

The human AIDS viruses--HIV-1 and HIV-2--impose major burdens on the health and economic status of many developing countries. Surveys of other animal species have revealed that related viruses--the SIVs are widespread in a large number of African simian primates where they do not appear to cause disease. Phylogenetic analyses indicate that these SIVs are the reservoirs for the human viruses, with SIVsm from the sooty mangabey monkey the most likely source of HIV-2, and SIVcpz from the common chimpanzee the progenitor population for HIV-1. Although it is clear that AIDS has a zoonotic origin, it is less certain when HIV-1 and HIV-2 first entered human populations and whether cross-species viral transmission is common among primates. Within infected individuals the process of HIV evolution takes the form of an arms race, with the virus continually fixing mutations by natural selection which allow it to escape from host immune responses. The arms race is less intense in SIV-infected monkeys, where a weaker immune response generates less selective pressure on the virus. Such a difference in virus-host interaction, along with a broadening of co-receptor usage such that HIV strains are able to infect cells with both CCR5 and CXCR4 chemokine receptors, may explain the increased virulence of HIV in humans compared to SIV in other primates.     

Isolation and antigenic characterization of human immunodeficiency virus (HIV) in Brazil

A retrovirus infecting a Brazilian AIDS patient was isolated and characterized in terms of its reactivity with sera from individuals infected with human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2). The Western blot analysis revealed that the Brazilian isolate is very similar to the well characterized HIV-1 strain. The serum of the patient from whom the virus was isolated did not react with the 140 kDa envelope glycoprotein specific for HIV-2.     

Filariasis and malaria sera from Orissa (India) lack human immunodeficiency virus (HIV) antibody reactivity

Reports exist indicating a correlation between seropositivity for human T-lymphotrophic virus (HTLV) antibodies and certain parasitic infections in some parts of the world. In 274 filariasis and 119 malarla sera examined from Orissa, none was reactive in a test for antibodies to human immunodeficiency virus (HIV).The author is with the AIDS Surveillance Unit, Regional Medical Research Centre (Indian Council of Medical Research), Bhubaneswar 751 016, India.     

Selective inhibition of human immunodeficiency virus (HIV) by 3'-azido-2', 3'-dideoxyguanosine in vitro

3'-Azido-2',3'-dideoxyguanosine (AzddGuo) is a potent and selective inhibitor of human immunodeficiency virus (HIV) in vitro. AzddGuo completely inhibits HIV-induced cytopathogenicity and viral antigen expression in MT-4 cells at a concentration of 5.0 microM. Its 50% effective dose for inhibiting HIV-induced cytopathogenicity is 1.4 microM, as compared to 6.4 microM for 2',3'-dideoxyadenosine (ddAdo). Thus, AzddGuo is approximately 4.6-fold more potent as an anti-HIV agent than ddAdo, one of the most promising compounds for the treatment of AIDS. However, AzddGuo is about 4.7 times more cytotoxic than ddAdo, so that its selectivity index, as based on the ratio of the 50% cytotoxic dose to the 50% antiviral effective dose, is almost the same as that of ddAdo (136 and 139, respectively).     

Immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic HIV protein

Elicitation of broad humoral immune responses is a critical factor in the development of effective HIV vaccines. In an effort to develop low-cost candidate vaccines based on multiepitopic recombinant proteins, this study has been undertaken to assess and characterize the immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic protein. This protein consists of V3 loops corresponding to five different HIV isolates, including MN, IIIB, RF, CC, and RU. In this study, both Escherichia coli and lettuce-derived C4(V3)6 have elicited local and systemic immune responses when orally administered to BALB/c mice. More importantly, lettuce-derived C4(V3)6 has shown a higher immunogenic potential than that of E. coli-derived C4(V3)6. Moreover, when reactivity of sera from mice immunized with C4(V3)6 are compared with those elicited by a chimeric protein carrying a single V3 sequence, broader responses have been observed. The lettuce-derived C4(V3)6 has elicited antibodies with positive reactivity against V3 loops from isolates MN, RF, and CC. In addition, splenocyte proliferation assays indicate that significant T-helper responses are induced by the C4(V3)6 immunogen. Taken together, these findings account for the observed elicitation of broader humoral responses by the C4(V3)6 multiepitopic protein. Moreover, they provide further validation for the production of multiepitopic vaccines in plant cells as this serves not only as a low-cost expression system, but also as an effective delivery vehicle for orally administered immunogens.     

Brucella abortus conjugated with a gp120 or V3 loop peptide derived from human immunodeficiency virus (HIV) type 1 induces neutralizing anti-HIV antibodies, and the V3-B. abortus conjugate is effective even after CD4+ T-cell depletion.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with loss of function and numbers of CD4+ T-helper cells. In order to bypass the requirement for CD4+ cells in antibody responses, we have utilized heat-inactivated Brucella abortus as a carrier. In this study we coupled a 14-mer V3 loop peptide (V3), which is homologous to 9 of 11 amino acids from the V3 loop of HIV-1 MN, and gp120 from HIV-1 SF2 to B. abortus [gp120(SF2)-B. abortus]. Our results showed that specific antibody responses, dominated by immunoglobulin G2a in BALB/c mice, were induced by these conjugates. Sera from the immunized mice bound native gp120 expressed on the surfaces of cells infected with a recombinant vaccinia virus gp160 vector (VPE16). Sera from mice immunized with gp120(SF2)-B. abortus inhibited binding of soluble CD4 to gp120, whereas sera from mice immunized with V3-B. abortus were ineffective. Sera from mice immunized with either conjugate were capable of blocking syncytium formation between CD4+ CEM cells and H9 cells chronically infected with the homologous virus. Sera from mice immunized with gp120(SF2)-B. abortus were more potent than sera from mice immunized with V3-B. abortus in inhibiting syncytia from heterologous HIV-1 laboratory strains. Importantly, in primary and secondary responses, V3-B. abortus evoked anti-HIV MN antibodies in mice depleted of CD4+ cells, and sera from these mice were able to inhibit syncytia. These findings indicate that B. abortus can provide carrier function for peptides and proteins from HIV-1 and suggest that they could be used for immunization of individuals with compromised CD4+ T-cell function.     

A reliable phenotype predictor for human immunodeficiency virus type 1 subtype C based on envelope V3 sequences

In human immunodeficiency virus type 1 (HIV-1) subtype B infections, the emergence of viruses able to use CXCR4 as a coreceptor is well documented and associated with accelerated CD4 decline and disease progression. However, in HIV-1 subtype C infections, responsible for more than 50% of global infections, CXCR4 usage is less common, even in individuals with advanced disease. A reliable phenotype prediction method based on genetic sequence analysis could provide a rapid and less expensive approach to identify possible CXCR4 variants and thus increase our understanding of subtype C coreceptor usage. For subtype B V3 loop sequences, genotypic predictors have been developed based on position-specific scoring matrices (PSSM). In this study, we apply this methodology to a training set of 279 subtype C sequences of known phenotypes (228 non-syncytium-inducing [NSI] CCR5⁺ and 51 SI CXCR4⁺ sequences) to derive a C-PSSM predictor. Specificity and sensitivity distributions were estimated by combining data set bootstrapping with leave-one-out cross-validation, with random sampling of single sequences from individuals on each bootstrap iteration. The C-PSSM had an estimated specificity of 94% (confidence interval [CI], 92% to 96%) and a sensitivity of 75% (CI, 68% to 82%), which is significantly more sensitive than predictions based on other methods, including a commonly used method based on the presence of positively charged residues (sensitivity, 47.8%). A specificity of 83% and a sensitivity of 83% were achieved with a validation set of 24 SI and 47 NSI unique subtype C sequences. The C-PSSM performs as well on subtype C V3 loops as existing subtype B-specific methods do on subtype B V3 loops. We present bioinformatic evidence that particular sites may influence coreceptor usage differently, depending on the subtype.