Apoptosis occurs predominantly in bystander cells and not in productively infected cells of HIV- and SIV-infected lymph nodes.

Although 13 years have passed since identification of human immunodeficiency virus-1 (HIV-1) as the cause of AIDS, we do not yet know how HIV kills its primary target, the T cell that carries the CD4 antigen. We and others have shown an increase in the percentage of apoptotic cells among circulating CD4+ (and CD8+) T cells of HIV-seropositive individuals and an increase in frequency of apoptosis with disease progression. However, it is not known if this apoptosis occurs in infected or uninfected T cells. We show here, using in situ labelling of lymph nodes from HIV-infected children and SIV-infected macaques, that apoptosis occurs predominantly in bystander cells and not in the productively infected cells themselves. These data have implications for pathogenesis and therapy, namely, arguing that rational drug therapy may involve combination agents targeting viral replication in infected cells and apoptosis of uninfected cells.

Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques.

A substantial risk in using live attenuated, multiply deleted viruses as vaccines against AIDS is their potential to induce AIDS. A mutant of the simian immunodeficiency virus (SIV) with large deletions in nef and vpr and in the negative regulatory element induced AIDS in six of eight infant macaques vaccinated orally or intravenously. Early signs of immune dysfunction were seen in the remaining two offspring. Prolonged follow-up of sixteen vaccinated adult macaques also showed resurgence of chronic viremia in four animals: two of these developed early signs of disease and one died of AIDS. We conclude that this multiply deleted SIV is pathogenic and that human AIDS vaccines built on similar prototypes may cause AIDS.

Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection.

Although maternal human immunodeficiency virus type 1 (HIV-1) transmission occurs during gestation, intrapartum and postpartum (by breast-feeding), 50-70% of all infected children seem to acquire HIV-1 shortly before or during delivery. Epidemiological evidence indicates that mucosal exposure is an important aspect of intrapartum HIV transmission. A simian immunodeficiency virus (SIV) macaque model has been developed that mimics the mucosal exposure that can occur during intrapartum HIV-1 transmission. To develop immunoprophylaxis against intrapartum HIV-1 transmission, we used SHIV-vpu+ (refs. 5,6), a chimeric simian-human virus that encodes the env gene of HIV-IIIB. Several combinations of human monoclonal antibodies against HIV-1 have been identified that neutralize SHIV-vpu+ completely in vitro through synergistic interaction. Here, we treated four pregnant macaques with a triple combination of the human IgG1 monoclonal antibodies F105, 2G12 and 2F5. All four macaques were protected against intravenous SHIV-vpu+ challenge after delivery. The infants received monoclonal antibodies after birth and were challenged orally with SHIV-vpu+ shortly thereafter. We found no evidence of infection in any infant during 6 months of follow-up. This demonstrates that IgG1 monoclonal antibodies protect against mucosal lentivirus challenge in neonates. We conclude that epitopes recognized by the three monoclonal antibodies are important determinants for achieving substantial protection, thus providing a rational basis for AIDS vaccine development.

Retroviruses and mouse embryos: a rapid model for neurovirulence and transplacental antiviral therapy.

A murine model in which neurotropic retroviral infection can be studied over short periods of time was developed. Microinjection of Cas-Br-E virus into midgestation mouse embryos caused paralysis and death within 25 days after birth, in contrast to virus-infected neonates which develop disease only after 4 months. To evaluate whether antiviral drugs could cross the placental barrier and influence the course of the disease, the drug 3'-azido-3'-deoxythymidine (AZT) was administered to infected embryos through the drinking water of pregnant females. AZT treatment markedly retarded the onset and course of virus-induced central nervous system disease, permitting animals to survive beyond 4 months of age. These results are evidence for effective antiviral treatment during gestation and in the perinatal period and are of potential significance for the management of maternal transmission of the acquired immune deficiency syndrome (AIDS) virus.

Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques.

Adult macaques do not develop disease after infection with a nef deletion mutant of the simian immunodeficiency virus (SIV) and are protected against challenge with pathogenic virus. This finding led to the proposal to use nef-deleted viruses as live, attenuated vaccines to prevent human acquired immunodeficiency syndrome (AIDS). In contrast, neonatal macaques developed persistently high levels of viremia after oral exposure to and SIV nef, vpr, and negative regulatory element (NRE) deletion mutant. Severe hemolytic anemia, thrombocytopenia, and CD4+ T cell depletion were observed, indicating that neither nef nor vpr determine pathogenicity in neonates. Because such constructs have retained their pathogenic potential, they should not be used as candidate live, attenuated virus vaccines against human AIDS.

Infection and AIDS in adult macaques after nontraumatic oral exposure to cell-free SIV.

Unprotected receptive anal intercourse is a well-recognized risk factor for infection with human immunodeficiency virus-type 1 (HIV-1). Isolated human case reports have implicated HIV-1 transmission by oral-genital exposure. Adult macaques exposed nontraumatically to cell-free simian immunodeficiency virus (SIV) through the oral route became infected and developed acquired immunodeficiency syndrome (AIDS). The minimal virus dose needed to achieve systemic infection after oral exposure was 6000 times lower than the minimal dose required to achieve systemic infection after rectal exposure. Thus, unprotected receptive oral intercourse, even in the absence of mucosal lesions, should be added to the list of risk behaviors for HIV-1 transmission.

Ribose metabolism and nucleic acid synthesis in normal and glucose-6-phosphate dehydrogenase-deficient human erythrocytes infected with Plasmodium falciparum.

The metabolism of pentose-phosphate was investigated in Plasmodium falciparum-infected normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient human red blood cells in vitro. 5'-Phosphoribosyl-1-pyrophosphate (PRPP) content of infected normal red blood cells was increased 50-60-fold at the parasite trophozoite growth stage over that of uninfected cells. The PRPP increment in infected G6PD-deficient cells at comparable stage and parasitemia was only 40% of the value in normal infected cells. Red blood cell PRPP synthetase activity did not change during the growth cycle of the parasite and was similar in both normal and G6PD-deficient cells. Reduced glutathione (GSH) content of G6PD-deficient cells under conditions of culture fell to low or undetectable levels. These low levels of GSH were shown to inhibit the function of red blood cell PRPP synthetase, which requires GSH for full activity. Measurements of the incorporation of 1-14C or 6-14C selectively labeled glucose into parasite nucleic acids revealed that in normal infected red cells, approximately 20% of the pentose was produced via the oxidation of glucose-6-phosphate, whereas in infected G6PD-deficient cells (Mediterranean type), none of the pentose was produced via the oxidative pathway. It is concluded that the low level of reduced GSH found in G6PD deficiency and the resultant partial inhibition of PRPP synthetase together with the missing oxidative pathway for ribose phosphate production can account fully for the reduced parasite growth rate in G6PD-deficient red blood cells described previously. Of these two mechanisms, the predominant one is the impaired PRPP synthetase activity due to low GSH levels in enzyme-deficient red blood cells. The contribution to the ribose-phosphate pool by the hexose monophosphate shunt is relatively minor. A co-existing oxidative stress (which is often hypothesized to mediate the destruction of parasitized red blood cells) is not required to explain growth inhibition in this scheme and does not represent the most straight-forward explanation of the data described in this report.

Murine models for evaluating antiretroviral therapy.

The pandemic of the acquired immunodeficiency syndrome (AIDS), caused by the human immunodeficiency virus type 1 (HIV-1), requires rapid development of effective therapy and prevention. Analysis of candidate anti-HIV-1 drugs in animals is problematic since no ideal animal model for HIV-1 infection and disease exists. For many reasons, including small size, availability of inbred strains, immunological reagents, and lymphokines, murine systems have been used for in vivo analysis of antiretroviral agents. Here we review currently available murine models involving HIV-1 in transgenic mice and in chimeric mice reconstituted with human cells, as well as murine systems using retroviruses of the subfamily Oncovirinae rather than Lentivirinae. We report our results on various antiretroviral treatment strategies, including chemoprophylaxis after acute retroviral exposure, therapy of chronic viremia, quantitative analysis of combination therapy, and therapy during pregnancy and in the neonatal period aimed at preventing viremia in the offspring. Due to our highly effective postexposure treatment protocols with 3'-azido-3'-deoxythymidine (zidovudine) combined with recombinant human interferon-alpha A/D, retrovirus-inoculated mice developed immunity to the virus to which they were exposed, which will allow us to determine the nature of protective antiretroviral immunity in inbred mice.

Antibodies: can they protect against HIV infection?

More than 20 million people have died since the discovery of human immunodeficiency virus (HIV), yet a broadly reactive AIDS vaccine remains elusive. Neutralizing antibody (nAb) response-based vaccine strategies were the first to be tested; however, when the difficulty in neutralizing primary HIV isolates was recognized, vaccine development focused instead on generating cytotoxic T-lymphocyte (CTL) responses. Recently, interest in anti-HIV nAbs has been revived by the impressive protection achieved in primates given passive immunization with neutralizing monoclonal antibodies (nmAbs) isolated from HIV clade B-infected individuals. The nmAbs used in these studies target conserved, functionally important epitopes in HIV gp120 and gp41. Regimens involving combinations of such human nmAbs or high-dose single-agent nmAb protected monkeys against intravenous (iv) and mucosal challenges with simian-human immunodeficiency virus (SHIV) strains encoding X4, X4R5 or R5 HIV env genes. In several such studies, sterilizing immunity was achieved, thus providing proof-of-concept that nAbs targeting conserved epitopes can be fully protective. The existence of these broadly reactive nmAbs suggests that it may be possible to design immunogens capable of inducing similar nAb responses by active vaccination. Unraveling the three-dimensional structures involved in the nmAb-HIV Env epitope interactions may facilitate the future development of a potent AIDS vaccine. This review is focused on the importance of nAbs in protecting against HIV infection or in containing viral spread, with particular emphasis on the successful use of nmAbs in passive immunization studies. The implications of the data from these studies on AIDS vaccine design in general are also discussed.

Murine and simian retrovirus models: the threshold hypothesis.

By considering the dynamic relationship between retroviruses and their hosts, we have developed a unifying hypothesis to explain such disparate clinical phenomena as differential pathogenicity of a given virus in adults and neonates, transient infection with clearance of provirus-containing cells, long-term non-progression and vaccine effects of fully pathogenic viruses. The threshold hypothesis predicts that an opportunity exists during acute retroviral infection to influence the ultimate clinical outcome: if virus replication is kept below threshold by any means, including drug therapy or passive immunoprophylaxis with neutralizing antibodies, the host will prevail and win the race.

In vivo analysis of castanospermine, a candidate antiretroviral agent.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine), an inhibitor of glycoprotein processing, has been shown to inhibit the human immunodeficiency virus type 1 (HIV-1) with acceptable toxicity in cultured cells. In contrast to reverse transcriptase inhibitors, castanospermine targets host enzymes. We have analyzed castanospermine in murine systems, using cultured cells as well as live animals. Plaque formation by Rauscher murine leukemia virus (RLV) was inhibited with a median inhibitory concentration (IC50) of 2 micrograms/ml. RLV-exposed BALB/c mice treated with a 20 day course of castanospermine starting 4 h postinoculation showed a dose-dependent inhibition of splenomegaly. Oral castanospermine therapy given to chronically RLV-infected mice prolonged median survival from 36 to 94 days when compared to untreated controls (p = 0.007). Castanospermine was better tolerated orally than intraperitoneally at the same dose. Toxic effects included weight loss, lethargy, and dose-dependent thrombocytopenia. At the highest intraperitoneal dose, lymphoid depletion occurred in thymus, spleen, and lymph nodes. We conclude that castanospermine is an active antiviral agent in animals and that prolonged oral administration is tolerable; however, when compared to 3'-azido-3'-deoxythymidine in the same murine system, castanospermine was less active and more toxic.

Postexposure chemoprophylaxis with ZDV or ZDV combined with interferon-alpha: failure after inoculating rhesus monkeys with a high dose of SIV.

Presently, no information is available regarding the efficacy of chemoprophylaxis in controlled human trials following accidental exposure to the human immunodeficiency virus (HIV). Using the closely related simian immunodeficiency virus (SIV) in rhesus monkeys, which develop a disease closely resembling human AIDS, we tested the efficacy of either single-agent 3'-azido-3'-deoxythymidine (ZDV) or the combination of ZDV plus recombinant human interferon-alpha (IFN-alpha). Treatment was started 3 h following inoculation of a high dose of SIV and continued for 21 days. SIV-inoculated control animals remained untreated. Virus was recovered from all monkeys on day 8, and by week 7 all had seroconverted. In contrast to monkeys treated with ZDV alone, animals given combination therapy had lower levels of p27 gag antigen compared to untreated controls on day 8 (p = 0.043). We conclude that neither treatment regimen could prevent infection after high-dose virus exposure; however, combination therapy may have depressed the level of virus replication more effectively than ZDV alone.

Castanospermine vs. its 6-O-butanoyl analog: a comparison of toxicity and antiviral activity in vitro and in vivo.

Inhibitors of glycoprotein processing, such as castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine), have been shown previously to inhibit human immunodeficiency virus type 1 (HIV-1) with acceptable toxicity in cultured human cells. In prior experiments, we have tested the toxicity and antiviral efficacy of castanospermine in mice infected with the Rauscher murine leukemia virus (RLV). When compared with 3'-azido-3'-deoxythymidine (AZT, zidovudine), castanospermine was less effective and more toxic. Since the 6-O-butanoyl analog of castanospermine was previously found to have a more favorable activity profile than the parent compound against HIV-1 in cultured cells, we compared the antiviral efficacy of both compounds in parallel in vitro and in vivo in the RLV system. Plaque formation in the XC assay was inhibited with a 50% inhibitory concentration (IC50) of 2.4 microM for the 6-O-butanoyl analog of castanospermine, as compared to 9 microM for castanospermine. For both compounds, concentrations resulting in significant cytotoxicity were about ten times higher. Both compounds significantly decreased HIV-1 env-induced syncytium formation in a novel in vitro assay. In RLV-exposed mice, the 6-O-butanoyl analog showed no advantage over the parent compound: both curves for toxicity as well as antiviral efficacy were super-imposable. We conclude that the 6-O-butanoyl analog of castanospermine as well as castanospermine itself are active antiviral agents in mice and that prolonged oral administration is tolerable. However, in comparison to AZT, their antiviral activity profiles are less favorable.

Transplacental antiretroviral therapy with 9-(2-phosphonylmethoxyethyl)adenine is embryotoxic in transgenic mice.

Transgenic Mov-14 mice, which carry the provirus of Moloney murine leukemia virus (Mo-MuLV) in the germ line and begin to produce infectious virus on embryonic day 14, were used to evaluate the ability of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to cross the placenta and protect embryos from viremia. We have used the Mov-14 model previously to demonstrate the antiviral efficacy and lack of teratogenicity of transplacental therapy with 3'-azido-3'-deoxythymidine (zidovudine, ZDV). PMEA was administered to pregnant females by daily intraperitoneal injection or by osmotic pump. In contrast to ZDV, PMEA was either noneffective in preventing viremia in the offspring or embryotoxic, depending on the dose. The specific toxic effects seen were resorption of pregnancy, low birth weight, and neonatal death. Histopathological analysis of neonatal mice exposed to PMEA showed severe lymphoid depletion of the thymus. We conclude that PMEA therapy is contraindicated for use during pregnancy.

Simian immunodeficiency virus infection via amniotic fluid: a model to study fetal immunopathogenesis and prophylaxis.

The rising prevalence of infection with the human immunodeficiency virus type 1 (HIV-1) in young women will increase the number of infected children worldwide. Because HIV-1 seems to be transmitted mostly intrapartum, fetal infection probably occurs mainly via skin or mucous membrane exposure. A model for this route of fetal infection has been established in primates. After injecting the simian immunodeficiency virus (SIV) into amniotic fluid during late gestation, six of seven rhesus monkeys were born infected. All infected neonates were viable and showed signs of disease, such as low birth weights, lymphadenopathy, and rashes. Cytotoxic T-cell responses to SIV were absent in neonates, but present in mothers. The high fetal infection rate allows studies of lentiviral immunopathogenesis during ontogeny and the development of strategies to prevent maternal HIV-1 transmission.

Interferon-alpha and 3'-azido-3'-deoxythymidine are highly synergistic in mice and prevent viremia after acute retrovirus exposure.

This study was undertaken to calculate the in vivo drug interactions between recombinant human interferon-alpha A/D (rHuIFN-alpha A/D) and 3'-azido-3'-deoxythymidine (AZT) in a quantitative model for retroviral viremia. When given as single agents, both AZT and rHuIFN-alpha A/D suppressed virus-induced splenomegaly in a dose-dependent fashion in mice inoculated with Rauscher murine leukemia virus (RLV). However, suppressive doses of single-agent AZT caused anemia after 20 days of therapy. Combining rHuIFN-alpha A/D with AZT allowed drastic dose reductions for each agent while maintaining greater than or equal to 93% inhibition of splenomegaly. No clinically significant toxicity was seen. Computer analysis with the isobologram technique and combination index method showed that these combination regimens were highly synergistic. A 20-day course of AZT + rHuIFN-alpha A/D started 4 h after virus exposure was protective against RLV viremia and disease. After cessation of therapy, the animals were resistant to rechallenge with fully infectious RLV. We conclude that prompt initiation of effective combination therapy after retroviral exposure prevented viremia and disease and led to protective immunity.

Synergy, activity and tolerability of zidovudine and interferon-alpha in patients with symptomatic HIV-1 infection: AIDS Clincal Trial Group 068.

Thirty-four subjects with symptomatic HIV-1 infection, p24 antigenaemia, and CD4 cell counts > 200/mm3 were randomly assigned to receive treatment with either zidovudine (ZDV) orally, interferon-alpha (IFN-alpha) subcutaneously, or both at respective low (200 mg ZDV/ 2 million international units IFN-alpha (MIU)), middle (400 mg/4 MIU) or high (600 mg/6 MIU) daily dose levels for 12 weeks. Thereafter, all patients received combination therapy at the initially assigned dose level to a total of 96 weeks. This design permitted analysis by the combination index (CI) method, which demonstrated antiretroviral synergy between ZDV and IFN-alpha with respect to p24 antigen suppression. Over the first 12 weeks, combination therapy was acceptably tolerated, more so than IFN-alpha monotherapy, and it was significantly more active in suppressing antigenaemia than either of the monotherapies. Similarly, the high-dose combination was the most active dose level over weeks 12 to 96. Combination ZDV/IFN-alpha at the optimal dose level defined by this trial merits further study. In addition, the CI design strategy employed here may be useful for the investigation of new antiretroviral combinations.

Synthesis of the 2-chloro analogues of 3'-deoxyadenosine, 2',3'-dideoxyadenosine, and 2',3'-didehydro-2',3'-dideoxyadenosine as potential antiviral agents.

2-Chloro-3'-deoxyadenosine (2-chlorocordycepin), 2-chloro-2',3'-dideoxyadenosine (2-ClddAdo), and 2-chloro-2',3'-didehydro-2',3'-dideoxyadenosine (2-ClddeAdo) were synthesized from 2-chloroadenosine (2-ClAdo) as candidate antiretroviral agents on the basis that 2-chloro substitution would prevent enzymatic deamination and increase efficacy relative to 2',3'-dideoxyadenosine (ddAdo). Reduction of 2-chloro-5'-(4,4'-dimethoxytrityl)-2',3'-O-thiocarbonyladenosine with n-Bu3SnH, followed by detritylation with AcOH, unexpectedly gave a mixture of 2-chlorocordycepin and 2-chloroadenine. Treatment of the crude n-Bu3SnH reduction product with 1,1'-thiocarbonyldiimidazole, followed by another cycle of n-Bu3SnH reduction and detritylation with silica gel afforded 2-ClddAdo and a byproduct identified as 2-chloro-2',3'-O-methyleneadenosine. Treatment of 2-chloro-5'-O-(4,4'-dimethoxytrityl)-2',3'-thiocarbonyladenosine with 1,3-dimethyl-2-phenyl-1,3,2-diazaphospholidine followed by silica gel detritylation afforded 2-ClddeAdo. 2-ClddAdo and 2-ClddeAdo were tested for activity against human immunodeficiency virus (HIV) in a cultured human T4+ lymphocyte cell line. At a concentration of 100 microM, 2-ClddAdo inhibited reverse transcriptase (RT) production by 97%, while 2',3'-dideoxyadenosine (ddAdo) gave greater than 99% inhibition. In growth assays against uninfected T4+ cells, however, 100 microM 2-ClddAdo gave 23% inhibition while 100 microM ddAdo was nontoxic. At a nontoxic concentration of 20 microM, RT production was 75% inhibited by ddAdo but only 43% inhibited by 2-ClddAdo. Thus, a 2-chloro substituent increased host cell toxicity but decreased antiretroviral activity. The unsaturated analogue 2-ClddeAdo was more cytotoxic than 2-ClddAdo, and antiviral effects could not be measured above 20 microM, where there was only 75% inhibition of RT production. Because of the decreased therapeutic index of 2-ClddeAdo relative to 2-ClddAdo and ddAdo, greater than 90% inhibition of viral protein synthesis at a noncytotoxic concentration could not be achieved. In growth assays with cultured human T and B lymphocytes, 100 microM 2-chlorocordycepin gave 60-70% growth inhibition, while the IC50 against mouse fibroblasts was only 30 microM. The high cytotoxicity of 2-chlorocordycepin precluded consideration of this compound as an antiviral agent.

One-step, highly efficient site-directed mutagenesis by toxic protein selection.

A fast and efficient site-directed mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plasmid. Once the target gene is cloned into pTPS19, desired mutations can be introduced with two primers. The first contains the desired mutation, and the second is designed to create a +1 frame shift in the ccdB gene to inactivate the CcdB protein. The mutants can be directly selected on LB plates containing IPTG, through which the toxic CcdB protein is induced, thereby eliminating cells carrying wild-type parental plasmids. Based on stringent selection through the toxic CcdB protein, mutagenesis efficiency of 90%-100% was reached even after one round of transformation.

Simultaneous detection of simian retrovirus type D serotypes 1, 2, and 3 by polymerase chain reaction.

Asymptomatic infection of macaques with macaques with simian retroviruses type D (SRV/D), the etiologic agents of one form of retrovirus-induced simian immunodeficiency disease, can confound experiments with the simian immunodeficiency virus (SIV), which also induces immunodeficiency disease in macaques. The SIV/macaque model is the preferred nonhuman primate model for AIDS-related research. Serological screening for SRV/D alone is insufficient because not all infected animals seroconvert, and virus isolation by cocultivation may require 4 to 6 weeks. We have established a DNA polymerase chain reaction (PCR) assay. One set of nested primers allows detection of SRV/D serotypes 1, 2, and 3 and distinguishes SRV-2 from the other two serotypes. The PCR assay is sensitive; a single proviral copy of SRV/D could be detected in 150,000 to 210,000 macaque peripheral blood mononuclear cells (PBMCs). When applied to a panel of virus isolation-positive macaque samples, the PCR assay was positive in 100% of the tests. No false-positive results were seen when known specific-pathogen-free (SPF) macaques were examined. We propose that macaques be screened with a combination of SRV/D serology and this DNA PCR assay prior to enrollment in experiments with SIV.