Late-Stage Derivatization of Oleanolic Acid-Based Anti-HIV-1 Compounds.

A 12-keto-type oleanolic acid derivative (4) has been identified as a potent anti-human immunodeficiency virus type-1 (HIV-1) compound that demonstrates synergistic effects with several types of HIV-1 neutralizing antibodies. In the present study, we used a common key synthetic intermediate to carry out the late-stage derivatization of an anti-HIV compound based on the chemical structure of a 12-keto-type oleanolic acid derivative. To execute this strategy, we designed a diketo-type oleanolic acid derivative (5) for chemoselective transformation, targeting the carboxy group and the hydroxyl group on the statine unit, as well as the 3-carbonyl group on the oleanolic acid unit, as orthogonal synthetic handles. We carried out four types of chemoselective transformations, leading to identification of the indole-type derivative (16) as a novel potent anti-HIV compound. In addition, further optimization of the ß-hydroxyl group on the statine unit provided the R-4-isobutyl γ-amino acid-type derivative (6), which exhibited potent anti-HIV activity comparable to that of 4 but with reduced cytotoxicity.

Mpox Neutralizing Antibody Response to LC16m8 Vaccine in Healthy Adults.

Mpox Neutralizing Antibody Response to LC16m8 VaccineIn this study of 50 healthy volunteers in Japan, a smallpox vaccine (LC16m8) exhibited a robust neutralizing antibody response against two strains of the mpox virus. With a 94% "take" rate by day 14, seroconversion rates on day 28 were 72 and 70% against the Zr599 and Liberia strains, respectively, decreasing to 30% for both on day 168; no serious adverse events occurred.

Plasmacytoid dendritic cells stimulated with Lactococcus lactis strain Plasma produce soluble factors to suppress SARS-CoV-2 replication.

Innate immune responses are important in the control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. We have previously found a lactic acid bacteria species, Lactococcus lactis strain Plasma (LC-Plasma), which possesses specific feature to activate plasmacytoid dendritic cells (pDCs) and thus may affect innate immune responses. Here, we investigated the impact of pDC activation by LC-Plasma on SARS-CoV-2 replication in vitro. Addition of the culture supernatant of pDCs stimulated with LC-Plasma resulted in suppression of SARS-CoV-2 replication in Vero and Calu-3 cells. We confirmed interferon-α (IFN-α) secretion in the supernatant of pDCs stimulated with LC-Plasma and induction of IFN-stimulated genes in cells treated with the pDC supernatant. Anti-IFN-α antibody impaired the suppression of SARS-CoV-2 replication by the supernatant of LC-Plasma-stimulated pDCs, suggesting that IFN-α plays an important role in the SARS-CoV-2 suppression. Our results indicate the potential of LC-Plasma to induce inhibitory responses against SARS-CoV-2 replication through pDC stimulation with IFN-α secretion.

Hybrids of small CD4 mimics and gp41-related peptides as dual-target HIV entry inhibitors.

Hybrid molecules containing small CD4 mimics and gp41-C-terminal heptad repeat (CHR)-related peptides have been developed. A YIR-821 derivative was adopted as a CD4 mimic, which inhibits the interaction of gp120 with CD4. SC-peptides, SC34 and SC22EK, were also used as CHR-related peptides, which inhibit the interaction between the N-terminal heptad repeat (NHR) and CHR and thereby membrane fusion. Therefore, these hybrid molecules have dual-targets of gp120 and gp41. In the synthesis of the hybrid molecules of CD4 mimic-SC-peptides with different lengths of linkers, two conjugating methods, Cu-catalyzed azide-alkyne cycloaddition and direct cysteine alkylation, were performed. The latter reaction caused simpler operation procedures and higher synthetic yields than the former. The synthesized hybrid molecules of CD4 mimic-SC22EK have significantly higher anti-HIV activity than each sole agent. The present data should be useful in the future design of anti-HIV agents as dual-target entry inhibitors.

Development of a novel Macaque-Tropic HIV-1 adapted to cynomolgus macaques.

Macaque-tropic HIV-1 (HIV-1mt) variants have been developed to establish preferable primate models that are advantageous in understanding HIV-1 infection pathogenesis and in assessing the preclinical efficacy of novel prevention/treatment strategies. We previously reported that a CXCR4-tropic HIV-1mt, MN4Rh-3, efficiently replicates in peripheral blood mononuclear cells (PBMCs) of cynomolgus macaques homozygous for TRIMCyp (CMsTC). However, the CMsTC challenged with MN4Rh-3 displayed low viral loads during the acute infection phase and subsequently exhibited short-term viremia. These virological phenotypes in vivo differed from those observed in most HIV-1-infected people. Therefore, further development of the HIV-1mt variant was needed. In this study, we first reconstructed the MN4Rh-3 clone to produce a CCR5-tropic HIV-1mt, AS38. In addition, serial in vivo passages allowed us to produce a highly adapted AS38-derived virus that exhibits high viral loads (up to approximately 106 copies ml-1) during the acute infection phase and prolonged periods of persistent viremia (lasting approximately 16 weeks postinfection) upon infection of CMsTC. Whole-genome sequencing of the viral genomes demonstrated that the emergence of a unique 15-nt deletion within the vif gene was associated with in vivo adaptation. The deletion resulted in a significant increase in Vpr protein expression but did not affect Vif-mediated antagonism of antiretroviral APOBEC3s, suggesting that Vpr is important for HIV-1mt adaptation to CMsTC. In summary, we developed a novel CCR5-tropic HIV-1mt that can induce high peak viral loads and long-term viremia and exhibits increased Vpr expression in CMsTC.

Association of envelope-specific B-cell differentiation and viral selective pressure signatures in HIV-1 CRF01_AE infection.

OBJECTIVE: In HIV type 1 (HIV-1) infection, virus-specific B-cell and neutralizing antibody (NAb) responses are impaired but exert selective pressure on target viral Envelope (Env) resulting in prominent sequence diversification among geographical areas. The basal induction patterns of HIV Env-specific B cells and their interaction with HIV Env awaits clarification. DESIGN: We investigated the relationship of Env polymorphisms and Env-specific B-cell responses in treatment-naive HIV-1 CRF01_AE-infected Vietnamese. METHODS: Samples of 43 HIV-1 CRF01_AE infection-identified individuals were divided into acute-phase ( n  = 12) and chronic-phase ( n  = 31) by combined criteria of serological recent-infection assay and clinical parameters. We quantified subcloning-based polymorphic residue site numbers in plasma-derived Env variable region 1-5 (V1-V5)-coding regions within each individual, designating their summation within each region as variant index. Peripheral blood Env gp 140-specific B-cell responses and plasma neutralizing activity of Env pseudoviruses were examined to analyze their relationship with variant index. RESULTS: HIV-1 CRF01_AE Env gp140-specific total B-cell and plasma cell (CD19 + IgD - CD27 + CD38 + CD138 + ) responses were determined. In chronic-phase samples, significant correlation of variant index in all Env V1-V5 regions with Env-specific plasma cell responses was shown, and V1-V5 total variant index correlated stronger with Env-specific plasma cell as compared with total Env-specific B-cell responses. Env V5 variant index was significantly higher in chronic-phase cross-neutralizers of V5-polymorphic/VRC01-insensitive CRF01_AE Env. CONCLUSION: Results revealed the association between circulating Env-specific plasma cell responses and Env polymorphisms, implicating selective pressure on Env by plasma cell-derived antibodies and conversely suggests that Env-specific B-cell induction alone is insufficient for exerting Env selective pressure in HIV infection.

Design, synthesis, and bio-evaluation of novel triterpenoid derivatives as anti-HIV-1 compounds.

Two betulinic acid derivatives, RPR103611 (2) and IC9564 (3) were previously reported to be potent HIV-1 entry inhibitors. In this current study, a SAR study of the triterpenoid moiety of 2 and 3 has been performed and an oleanolic acid derivative (4) was identified as a novel HIV-1 entry inhibitor. In addition, the combination of 4 with several-type of HIV-1 neutralizing antibodies provided significant synergistic effects. The synthetic utility of the CC double bond in the C-ring of 4 was also demonstrated to develop the 12-keto-type oleanolic acid derivative (5) as a potent anti-HIV compound. This simple transformation led to a significantly increased anti-HIV activity and a reduced cytotoxicity of the compound.

Neutralizing-antibody-independent SARS-CoV-2 control correlated with intranasal-vaccine-induced CD8+ T cell responses.

Effective vaccines are essential for the control of the coronavirus disease 2019 (COVID-19) pandemic. Currently developed vaccines inducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigate the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Seven vaccinated macaques exhibit significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared with nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs is significantly correlated with vaccine-induced, viral-antigen-specific CD8+ T cell responses. Our results indicate that CD8+ T cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8+ T cell responses to contribute to COVID-19 containment.

Exploratory studies on soluble small molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics, which inhibit the interaction of gp120 with CD4, have been developed. Original CD4 mimics such as NBD-556, which has an aromatic ring, an oxalamide linker and a piperidine moiety, possess significant anti-HIV activity but with their hydrophobic aromatic ring-containing structures are poorly soluble in water. We have developed derivatives with a halopyridinyl group in place of the phenyl group, such as KKN-134, and found them to have excellent aqueous solubility. Other leads that were examined are YIR-821, a compound with a cyclohexane group in a spiro attachment to a piperidine ring and a guanidino group on the piperidine nitrogen atom, and its PEGylated derivative, TKB-002. YIR-821 and TKB-002 retain potent anti-HIV activity. Here, new CD4 mimics, in which the phenyl group was replaced by a halopyridinyl group with the halogen atoms in different positions, their derivatives without a cyclohexane group on the piperidine ring and their hybrid molecules with PEG units were designed and synthesized. Some of these compounds show significantly higher aqueous solubility with maintenance of certain levels of anti-HIV activity. The present data should be useful in the future design of CD4 mimic molecules.

Subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques.

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure.

Hybrids of Small-Molecule CD4 Mimics with Polyethylene Glycol Units as HIV Entry Inhibitors.

CD4 mimics are small molecules that inhibit the interaction of gp120 with CD4. We have developed several CD4 mimics. Herein, hybrid molecules consisting of CD4 mimics with a long alkyl chain or a PEG unit attached through a self-cleavable linker were synthesized. In anti-HIV activity, modification with a PEG unit appeared to be more suitable than modification with a long alkyl chain. Thus, hybrid molecules of CD4 mimics, with PEG units attached through an uncleavable linker, were developed and showed high anti-HIV activity and low cytotoxicity. In investigation of pharmacokinetics in a rhesus macaque, a hybrid compound had a more effective PK profile than that of the parent compound, and intramuscular injection was a more useful administration route to maintain the high blood concentration of the CD4 mimic than intravenous injection. The presented hybrid molecules of CD4 mimics with a PEG unit would be practically useful when combined with a neutralizing antibody.

Activity and structural analysis of GRL-117C: a novel small molecule CCR5 inhibitor active against R5-tropic HIV-1s.

CCR5 is a member of the G-protein coupled receptor family that serves as an essential co-receptor for cellular entry of R5-tropic HIV-1, and is a validated target for therapeutics against HIV-1 infections. In the present study, we designed and synthesized a series of novel small CCR5 inhibitors and evaluated their antiviral activity. GRL-117C inhibited the replication of wild-type R5-HIV-1 with a sub-nanomolar IC50 value. These derivatives retained activity against vicriviroc-resistant HIV-1s, but did not show activity against maraviroc (MVC)-resistant HIV-1. Structural modeling indicated that the binding of compounds to CCR5 occurs in the hydrophobic cavity of CCR5 under the second extracellular loop, and amino acids critical for their binding were almost similar with those of MVC, which explains viral cross-resistance with MVC. On the other hand, one derivative, GRL-10018C, less potent against HIV-1, but more potent in inhibiting CC-chemokine binding, occupied the upper region of the binding cavity with its bis-THF moiety, presumably causing greater steric hindrance with CC-chemokines. Recent studies have shown additional unique features of certain CCR5 inhibitors such as immunomodulating properties and HIV-1 latency reversal properties, and thus, continuous efforts in developing new CCR5 inhibitors with unique binding profiles is necessary.

Soluble-type small-molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics have been reported previously as HIV-1 entry inhibitors, which block the interaction between the Phe43 cavity of HIV-1 gp120 and the host CD4. Known CD4 mimics such as NBD-556 possess significant anti-HIV activity but are less soluble in water, perhaps due to their hydrophobic aromatic ring-containing structures. Compounds with a pyridinyl group in place of the phenyl group in these molecules have been designed and synthesized in an attempt to increase the hydrophilicity. Some of these new CD4 mimics, containing a tetramethylpiperidine ring show significantly higher water solubility than NBD-556 and have high anti-HIV activity and synergistic anti-HIV activity with a neutralizing antibody. The CD4 mimic that has a cyclohexylpiperidine ring and a 6-fluoropyridin-3-yl ring has high anti-HIV activity and no significant cytotoxicity. The present results will be useful in the future design and development of novel soluble-type molecule CD4 mimics.

Isolation and structure determination of a new lasso peptide specialicin based on genome mining.

Based on genome mining, a new lasso peptide specialicin was isolated from the extract of Streptomyces specialis. The structure of specialicin was established by ESI-MS and NMR analyses to be a lasso peptide with the length of 21 amino acids, containing an isopeptide bond and two disulfide bonds in the molecule. The stereochemistries of the constituent amino acids except for Trp were determined to be L and the stereochemistry of Trp at C-terminus was determined to be D. Three dimensional structure of specialicin was determined based on NOE experimental data, which indicated that specialicin possessed the similar conformational structure with siamycin I. Specialicin showed the antibacterial activity against Micrococcus luteus and the moderate anti-HIV activity against HIV-1 NL4-3. The biosynthetic gene cluster of specialicin was proposed from the genome sequence data of S. specialis.

Flexibility of small molecular CD4 mimics as HIV entry inhibitors.

CD4 mimics such as YIR-821 and its derivatives are small molecules which inhibit the interaction between the Phe43 cavity of HIV-1 gp120 with host CD4, an interaction that is involved in the entry of HIV to cells. Known CD4 mimics generally possess three structural features, an aromatic ring, an oxalamide linker and a piperidine moiety. We have shown previously that introduction of a cyclohexyl group and a guanidine group into the piperidine moiety and a fluorine atom at the meta-position of the aromatic ring leads to a significant increase in the anti-HIV activity. In the current study, the effects of conformational flexibility were investigated by introduction of an indole-type group in the junction between the oxalamide linker and the aromatic moiety or by replacement of the oxalamide linker with a glycine linker. This led to the development of compounds with high anti-HIV activity, showing the importance of the junction region for the expression of high anti-HIV activity. The present data are expected to be useful in the future design of novel CD4 mimic molecules.

[Clinical Significance of Circulating Tumor DNA and Its Future Prospects].

"Liquid biopsy"is a diagnostic medium to detect very small amount of tumor-derived molecules in blood for selecting precise clinical treatments and giving a diagnosis. This article focuses on circulating tumor DNA(ctDNA)and describes its features, clinical significance as well as future prospects with recent findings in this field. "Liquid biopsy"using ctDNAis facilitated by recent developments of highly sensitive technologies, including digital PCR. "BEAMing"is a digital PCR method that can detect rare mutations in ctDNAand will be introduced with some clinical evidences in this article. ctDNAis tumorderived fragmented DNAthat is circulating freely in the blood of a cancer patient. Tests for ctDNAare less invasive, easier than tissue-based tests to collect specimens and can be used repeatedly regardless of any cancer types. Emerging clinical evidences indicate that ctDNAtests show a high concordance rate with tissue-based tests in the identification of oncogene genotypes and can be used for real-time monitoring in vivo, such as prediction of clinical treatments, recurrence and drug resistance. "Liquid biopsy" using ctDNAcontinues to develop, but is promising to complement conventional tissue-based tests. Clinical applications of ctDNAtests are expected to expand as a useful diagnostic tools in areas of therapy selection and monitoring for drug resistance.

Driving HIV-1 into a Vulnerable Corner by Taking Advantage of Viral Adaptation and Evolution.

Anti-retroviral therapy (ART) is crucial for controlling human immunodeficiency virus type-1 (HIV-1) infection. Recently, progress in identifying and characterizing highly potent broadly neutralizing antibodies has provided valuable templates for HIV-1 therapy and vaccine design. Nevertheless, HIV-1, like many RNA viruses, exhibits genetically diverse populations known as quasispecies. Evolution of quasispecies can occur rapidly in response to selective pressures, such as that exerted by ART and the immune system. Hence, rapid viral evolution leading to drug resistance and/or immune evasion is a significant barrier to the development of effective HIV-1 treatments and vaccines. Here, we describe our recent investigations into evolutionary pressure exerted by anti-retroviral drugs and monoclonal neutralizing antibodies (NAbs) on HIV-1 envelope sequences. We also discuss sensitivities of HIV-1 escape mutants to maraviroc, a CCR5 inhibitor, and HIV-1 sensitized to NAbs by small-molecule CD4-mimetic compounds. These studies help to develop an understanding of viral evolution and escape from both anti-retroviral drugs and the immune system, and also provide fundamental insights into the combined use of NAbs and entry inhibitors. These findings of the adaptation and evolution of HIV in response to drug and immune pressure will inform the development of more effective antiviral therapeutic strategies.

Biphasic CD8+ T-Cell Defense in Simian Immunodeficiency Virus Control by Acute-Phase Passive Neutralizing Antibody Immunization.

UNLABELLED: Identifying human immunodeficiency virus type 1 (HIV-1) control mechanisms by neutralizing antibodies (NAbs) is critical for anti-HIV-1 strategies. Recent in vivo studies on animals infected with simian immunodeficiency virus (SIV) and related viruses have shown the efficacy of postinfection NAb passive immunization for viremia reduction, and one suggested mechanism is its occurrence through modulation of cellular immune responses. Here, we describe SIV control in macaques showing biphasic CD8(+) cytotoxic T lymphocyte (CTL) responses following acute-phase NAb passive immunization. Analysis of four SIVmac239-infected rhesus macaque pairs matched with major histocompatibility complex class I haplotypes found that counterparts receiving day 7 anti-SIV polyclonal NAb infusion all suppressed viremia for up to 2 years without accumulating viral CTL escape mutations. In the first phase of primary viremia control attainment, CD8(+) cells had high capacities to suppress SIVs carrying CTL escape mutations. Conversely, in the second, sustained phase of SIV control, CTL responses converged on a pattern of immunodominant CTL preservation. During this sustained phase of viral control, SIV epitope-specific CTLs showed retention of phosphorylated extracellular signal-related kinase (ERK)(hi)/phosphorylated AMP-activated protein kinase (AMPK)(lo) subpopulations, implying their correlation with SIV control. The results suggest that virus-specific CTLs functionally boosted by acute-phase NAbs may drive robust AIDS virus control. IMPORTANCE: In early HIV infection, NAb responses are lacking and CTL responses are insufficient, which leads to viral persistence. Hence, it is important to identify immune responses that can successfully control such HIV replication. Here, we show that monkeys receiving NAb passive immunization in early SIV infection strictly control viral replication for years. Passive infusion of NAbs with CTL cross-priming capacity resulted in induction of functionally boosted early CTL responses showing enhanced suppression of CTL escape mutant virus replication. Accordingly, the NAb-infused animals did not show accumulation of viral CTL escape mutations during sustained SIV control, and immunodominant CTL responses were preserved. This early functional augmentation of CTLs by NAbs provides key insights into the design of lasting and viral escape mutation-free protective immunity against HIV-1 infection.

A novel dual luciferase assay for the simultaneous monitoring of HIV infection and cell viability.

Human immunodeficiency virus type 1 (HIV-1) reporter cell lines are critical tools for drug development. However, one disadvantage of HIV-1 reporter cell lines is that reductions in reporter gene activity need to be normalized to cytotoxicity, i.e., live cell numbers. Here, we developed a dual luciferase assay based on a R. reniformis luciferase (hRLuc)-expressing R5-type HIV-1 (NLAD8-hRLuc) and a CEM cell line expressing CCR5 and firefly luciferase (R5CEM-FiLuc). The NLAD8-hRLuc reporter virus was replication competent in peripheral blood mononuclear cells. The level of hRLuc was correlated with p24 antigen levels (p<0.001, R=0.862). The target cell line, R5CEM-FiLuc, stably expressed the firefly luciferase (FiLuc) reporter gene and allowed the simultaneous monitoring of compound cytotoxicity. The dual reporter assay combining a NLAD8-hRLuc virus with R5CEM-FiLuc cells permitted the accurate determination of drug susceptibility for entry, reverse transcriptase, integrase, and protease inhibitors at different multiplicities of infection. This dual reporter assay provides a rapid and direct method for the simultaneous monitoring of HIV infection and cell viability.

Small-Molecule CD4 Mimics Containing Mono-cyclohexyl Moieties as HIV Entry Inhibitors.

CD4 mimics are small molecules that inhibit the protein-protein interaction between gp120 and CD4, which is a key interaction for the entry of human immunodeficiency virus (HIV) into host immune cells. In the present study, mono-cyclohexyl-type CD4 mimics were designed to form hydrophobic and electrostatic interactions with Val430 and Asp368 located in the entrance of the Phe43 cavity of gp120, the interaction site of CD4. YIR-329, a novel 1-azaspiro[5.5]undecane derivative with a cyclohexyl ring attached to the piperidine ring, exhibited only slightly weaker anti-HIV activity than a previously described lead HAR-171, and modeling results indicated the formation of advantageous interactions by the para-chlorophenyl moiety of YIR-329. To introduce an electrostatic interaction with Asp368, derivatives with a guanidino group on the piperidine nitrogen atom were synthesized. Mono-cyclohexyl-type CD4 mimics with a guanidino group, such as YIR-819 (N(1) -(4-chlorophenyl)-N(2) -(1-(2-(N-(amidino)glycinamide)ethyl)-2-cyclohexylpiperidin-4-yl)oxalamide) and YIR-821 (1-(2-(5-guanidinovaleramide)ethyl derivative of YIR-819), were identified that exhibit approximately fivefold more potent anti-HIV activity than YIR-329. In combination with a neutralizing antibody, their anti-HIV activities were augmenting. Modeling results suggest that these compounds interact effectively with Val430 and either Asp368 or Asp474 in the gp120 Phe43 cavity. YIR-819 and YIR-821 represent useful lead compounds for the further development of HIV-1 entry inhibitors and could potentially be useful for co-administration with neutralizing antibodies for the treatment of HIV infection and AIDS.