Han G. Huisman

Persistent HIV antigenaemia and decline of HIV core antibodies associated with transition to AIDS.

Sequential serum samples from 13 homosexual men who seroconverted for antibodies to human immunodeficiency virus (HIV) were tested for HIV antigen. In one of these men, who developed the acquired immune deficiency syndrome (AIDS), HIV antigenaemia preceded the onset of AIDS by more than a year and persisted throughout the course of the disease. This antigenaemia was accompanied by the disappearance of IgG antibody reactivity to the major HIV core protein p24. In none of the 12 others, who all remained without serious disease, were serum concentrations of HIV antigen detected, except on one occasion in one man. All their serum samples showed strong IgG antibody reactivity to p24. Nine children who were infected with HIV in 1981 by plasma transfusion from a single donor were also followed up for HIV antigenaemia. HIV antigen was almost constantly present in the serum (26 of 28 samples) of five children who developed AIDS related complex or AIDS and less often in the serum (four of 10 samples) of four children who remained free of symptoms. The two children who developed AIDS showed a virtual absence of antibody reactivity to p24. These results indicate that increased HIV gene expression is a contributing factor to the development of AIDS and also provide evidence for a switch from latent to active HIV infection.

Proliferation-dependent HIV-1 infection of monocytes occurs during differentiation into macrophages.

Requirements for the establishment of productive infection with the human immunodeficiency virus type 1 (HIV-1) in primary monocytes were investigated. In vitro, monocytes rendered susceptible for infection after at least a 2-d culture, but when cultured in the presence of differentiation-inducing agent IL-4, accelerated susceptibility was seen. Complete resistance to HIV-1 infection was observed in monocytes that had been treated for 5 d with rIL-4, and comparable results were obtained with other differentiation inducers such as dexamethasone or 1,25(OH)2 vitamin D3 (1,25(OH)2vitD3). The inhibition of productive infection was not caused by downregulation of CD4 expression or HIV-1 transcription, nor by intracellular accumulation of virions. Since treatment with rIL-4, dexamethasone, or 1,25(OH)2vitD3 also resulted in complete inhibition of monocyte proliferation, we studied whether establishment of productive infection in monocytes is proliferation dependent. Irradiation or mitomycin-C treatment within 24 h after inoculation prevented productive HIV-1 infection of monocytes, suggesting a proliferation-dependent step early in the virus replication cycle. Polymerase chain reaction (PCR) analysis revealed the presence of only incomplete proviral DNA species in non-proliferating monocytes, indicating restriction of viral replication at the level of reverse transcription. Thus, in analogy with HIV-1 infection of CD4+ T cells, proliferation of monocytes during differentiation into macrophages is a prerequisite for productive infection with HIV.

Qualitative and quantitative detection of HIV-1 RNA by nucleic acid sequence-based amplification.

AimTo develop a method to detect HIV-1 viral RNA by amplifying a specific nucleic acid sequence. MethodThe nucleic acid sequence-based amplification (NASBA) method uses the simultaneous activity of avian myeloblastosis virus reverse transcriptase, T7 RNA polymerase and RNase H to amplify a specific nucleic acid target sequence. ValidationAn in vitro cultured HIV-1 stock solution was used to validate the NASBA method and determine the variation in RNA measurement. ConclusionAlthough NASBA is theoretically capable of specific amplification of RNA or DNA, it is most suitable for amplification of RNA, and therefore for detection of HIV-1 viral RNA.

HIV-1 macrophage tropism is determined at multiple levels of the viral replication cycle.

The ability of HIV-1 to infect macrophages is thought to be essential in AIDS pathogenesis. We tested the ability of 19 primary virus isolates to infect monocyte-derived macrophages (MDM) from different donors. Two HIV-1 isolates were able to establish a productive infection in MDM from all donors tested, whereas eight completely lacked this capacity. Next to these isolates with extreme phenotypes, 50% of the primary isolates under study displayed an intermediate phenotype. These intermediate macrophage-tropic isolates established a productive infection in MDM from some but not all donors tested. PCR analysis demonstrated that the capacity to replicate in MDM could be determined at the previously described level of virus entry. However, for intermediate macrophage-tropic isolates replication was abrogated at the level of reverse transcription. Entry of highly macrophage-tropic isolates resulted in efficient completion of the reverse transcription process, whereas entry of intermediate macrophage-tropic isolates did not. Our experiments indicate that primary HIV-1 isolates may differ in their dependency on cellular factors required for reverse transcription in MDM. Differences in susceptibility of MDM for in vitro HIV-1 infection suggest variation in the availability of these cellular factors between MDM from different individuals.

Use of competitive polymerase chain reaction to determine HIV-1 levels in response to antiviral treatments.

ObjectiveTo develop a competitive polymerase chain reaction technique with which to evaluate the usefulness of HIV-1 level as a marker of response to antiviral treatment. DesignHIV-1 sequences were assessed by competitive polymerase chain reaction in four subjects participating in a double-blind study of monotherapy versus combination therapy with nucleoside analogues. MethodsWe inserted a mutant construct of the HIV-1 pol sequence into a commercial vector, enabling us to generate known amounts of mutant DNA and RNA for competitive polymerase chain reaction. To measure HIV-1 DNA copies in cells, the mutant DNA fragments were allowed to compete in a 10-fold dilution series with a constant amount of nucleic acid from the subject. To measure HIV-1 RNA copies in plasma, in vitro synthesized mutant RNA was added in a 10-fold dilutation series to a constant amount of subject RNA and copy DNA was synthesized. DNA and copy DNA were used as the input for nested pol polymerase chain reaction. Mutant and wild-type amplimers were discriminated by size. ResultsThe competitive polymerase chain reaction technique has been validated in model experiments and can be used over a broad range (at least 6 logs) of levels. Three of the four subjects showed a decline of 1 log in proviral DNA levels in cells after beginning antiviral treatment. All four showed a decline of at least 1 log in viral RNA levels in plasma, but this decline was transient in one subject. ConclusionThe HIV-1 sequence level is a useful marker in antiviral treatment studies.

Evaluation of Three Second‐Generation and Three Confirmatory Assays for Antibodies to Human Immunodeficiency Virus

Abstract. The second‐generation enzyme immunoassays (EIAs) for antibodies against human immunodeficiency virus (HIV) from three manufacturers (Abbott, Organon, Wellcome) and three anti‐HIV confirmatory tests, i.e. Western Blot (WB, Biotech, Dupont), radioimmunoprecipitation assay (RIPA, CLB) and a competitive immunoassay (CIA, Abbott) were evaluated on a panel of 6,488 serum samples, which had previously been used for the comparison of seven first‐generation EIAs. For the present study the panel was expanded with sequential serum samples from 12 individuals followed at 1 ‐ to 3‐month intervals during seroconversion for anti‐HIV. The second‐generation EIAs and confirmatory tests were significantly more sensitive than the first‐generation EIAs as was demonstrated by detection of 10‐ to 100‐fold higher endpoint titers in anti‐HIV‐positive sera as well as by earlier detection of anti‐HIV in 7–11 of the 12 subjects, who seroconverted. In all sera obtained during early HIV infection anti‐gp160/120envantibodies (WB, CIA) were found in addition to anti‐p24 (WB, RIP A) and in serial twofold dilutions of these ‘seroconversion samples’ the new Abbott EIA and RIPA were significantly more sensitive than WB (p < 0.05), whereas CIA and the new Organon EIA were significantly less sensitive than WB (p < 0.05). The new Wellcome EIA was not statistically less sensitive than WB. The CIA was as sensitive as WB for antibodies to envelope proteins (gp 41, gp 160, gp 120), but considerably less sensitive for core (p24) antibodies, as was shown in sera obtained during early as well as later clinical stages of HIV infection. The frequencies of false‐positive reactions by the second‐generation EIAs on a panel of 292 tricky samples from patients with e.g. autoimmune diseases and acute viral infections and in a panel of 283 selected donor sera, with false‐positive reactivities in the first‐generation EIAs were: Abbott 1.0%, 0.3%, Wellcome 0%, 0%, and Organon 0.3%, 0%. By the confirmatory tests they were: WB (anti‐p24) 1.7%, 10.2%; RIPA (anti‐p24) 0.3%, 1.1%; and CIA 0.3%, 0.3%. It is concluded that application of highly sensitive second‐generation EIAs in blood banks is important to further reduce the risk of posttransfusion HIV infection.

Increased neutralization sensitivity and reduced replicative capacity of human immunodeficiency virus type 1 after short-term in vivo or in vitro passage through chimpanzees.

ABSTRACT Development of disease is extremely rare in chimpanzees when inoculated with either T-cell-line-adapted neutralization-sensitive or primary human immunodeficiency virus type 1 (HIV-1), at first excluding a role for HIV-1 neutralization sensitivity in the clinical course of infection. Interestingly, we observed that short-term in vivo and in vitro passage of primary HIV-1 isolates through chimpanzee peripheral blood mononuclear cells (PBMC) resulted in a neutralization-sensitive phenotype. Furthermore, an HIV-1 variant reisolated from a chimpanzee 10 years after experimental infection was still sensitive to neutralization by soluble CD4, the CD4 binding site recognizing antibody IgG1b12 and autologous chimpanzee serum samples, but had become relatively resistant to neutralization by polyclonal human sera and neutralizing monoclonal antibodies. The initial adaptation of HIV-1 to replicate in chimpanzee PBMC seemed to coincide with a selection for viruses with low replicative kinetics. Neither coreceptor usage nor the expression level of CD4, CCR5, or CXCR4 on chimpanzee PBMC compared to human cells could explain the phenotypic changes observed in these chimpanzee-passaged viruses. Our data suggest that the increased neutralization sensitivity of HIV-1 after replication in chimpanzee cells may in part contribute to the long-term asymptomatic HIV-1 infection in experimentally infected chimpanzees.

Mechanism of anti-HIV activity of succinylated human serum albumin

In the present study, we described the interaction of succinylated human serum albumin (Suc-HSA), a negatively charged anti-HIV-1 active protein, with HIV-1 gp120 and in detail with the third variable domain of gp120 (V3 loop). To this end, different assay formats were tested in which gp120- and V3-related peptides were presented in various configurations in order to investigate the effect of the conformational structure of the V3 loop on the interaction with negatively charged albumins. When gp120 presented via a lectin was used, it was observed that Suc-HSA bound to native gp120. The binding site appeared to be located at or near the thrombin digestion site (GPGRAF sequence) in the V3 loop of gp120, since the cleavage of the loop resulted in decreased binding of Suc-HSA. In addition, Suc-HSA was able to protect the V3 region of gp120 from cleaving with thrombin. In contrast, significant binding of Suc-HSA to V3 loop or gp120 peptides was not observed when both were presented in a fluid phase system, suggesting the involvement of a monovalent-low affinity binding of Suc-HSA. Using overlapping peptides delineating the whole V3 loop immobilized to CNBr-Sepharose, we noticed that the interaction of the V3 loop with Suc-HSA was predominantly induced by electrostatic interactions between positively charged linearized peptide fragments and Suc-HSA and was positively influenced by the presence of hydrophobic amino in the V3 loop fragments as well. Moreover, the highest affinity site was located at sites near the GPGRAF sequence. These observations add to the evidence, collected earlier, that Suc-HSA interferes at the level of virus entry, independent of interaction with the CD4 receptor. Since the recently discovered chemokine receptors are negatively charged, we can hypothesize that Suc-HSA is able to prevent the positively charged V3 loop from interacting with these types of receptors, thereby inhibiting virus entry.

Thermal Inactivation of Human Immunodeficiency Virus in Lyophilised Blood Products Evaluated by ID50 Titrations

Abstract. Inactivation of human immunodeficiency virus (HIV) in lyophilised small pool cryoprecipitate, factor VIII concentrate, prothrombin complex and C1 ‐esterase inhibitor concentrate by prolonged heat treatment (72 h, 60° C) was studied. Plasma products, inoculated prior to lyophilisation, had infectious titres ranging from 107 to 1010.5. Residual infectivity (TCID50) was assessed by multiple titrations on H9 cells in a macro system and subsequent detection of virus replication by determining reverse transcriptase activity. Kinetics of inactivation showed a biphasic pattern: during the first 8 h a variable TCID50 reduction up to 104.3 was observed, followed by an additional loss of 101–102.7 during the next 64 h. Heat treatment for 72 h resulted in a mean TCID50 reduction of 105. It is concluded that prolonged heat treatment may lead to the adequate prevention of HIV transmission by lyophilised plasma products.

Novel strategy for the selection of human recombinant Fab fragments to membrane proteins from a phage-display library.

Traditionally, the selection of phage-display libraries is performed on purified antigens (Ags), immobilized to a solid substrate. However, this approach may not be applicable for some Ags, such as membrane proteins, which for structural integrity strongly rely on their native environment. Here we describe an approach for the selection of phage-libraries against membrane proteins. The envelope glycoproteins (Env) of the Human Immunodeficiency Virus type-1 (HIV-1) were used as a model for a type-1 integral membrane protein. HIV-1IHI Env, expressed on the surface of Rabbit Kidney cells (RK13) with a recombinant vaccinia virus (rVV), was solubilized using the non-ionic detergent n-Octyl beta-D-glucopyranoside (OG). Membrane associated Env was reconstituted into vesicles by the simultaneous removal of detergent and free monomeric Env subunits by gel-filtration. The resulting antigen preparation, termed OG-P1IHI, was captured on microtiter plates coated with Galanthus nivalis agglutinin (GNA) and used for rounds of selection (panning) of a well-characterized phage-display library derived from an HIV-1 seropositive donor. Simultaneously, an identical experiment was performed with OG-P1IHI vesicles disrupted by Nonidet P-40 (NP-P1IHI). Both membrane-associated and soluble Ags were selected for vaccinia-specific clones (OG-P1IHI: 59/75 and NP-P1IHI: 1/75) and HIV-1-specific clones (OG-P1IHI: 11/75 and NP-P1IHI: 65/75) using our approach. Hence, the novel panning strategy described here may be applicable for selection of phage-libraries against membrane proteins.