Molecularly cloned SHIV-1157ipd3N4: a highly replication- competent, mucosally transmissible R5 simian-human immunodeficiency virus encoding HIV clade C Env.

Human immunodeficiency virus type 1 (HIV-1) clade C causes >50% of all HIV infections worldwide, and an estimated 90% of all transmissions occur mucosally with R5 strains. A pathogenic R5 simian-human immunodeficiency virus (SHIV) encoding HIV clade C env is highly desirable to evaluate candidate AIDS vaccines in nonhuman primates. To this end, we generated SHIV-1157i, a molecular clone from a Zambian infant isolate that carries HIV clade C env. SHIV-1157i was adapted by serial passage in five monkeys, three of which developed peripheral CD4(+) T-cell depletion. After the first inoculated monkey developed AIDS at week 137 postinoculation, transfer of its infected blood to a naïve animal induced memory T-cell depletion and thrombocytopenia within 3 months in the recipient. In parallel, genomic DNA from the blood donor was amplified to generate the late proviral clone SHIV-1157ipd3. To increase the replicative capacity of SHIV-1157ipd3, an extra NF-kappaB binding site was engineered into its 3' long terminal repeat, giving rise to SHIV-1157ipd3N4. This virus was exclusively R5 tropic and replicated more potently in rhesus peripheral blood mononuclear cells than SHIV-1157ipd3 in the presence of tumor necrosis factor alpha. Rhesus macaques of Indian and Chinese origin were next inoculated intrarectally with SHIV-1157ipd3N4; this virus replicated vigorously in both sets of monkeys. We conclude that SHIV-1157ipd3N4 is a highly replication-competent, mucosally transmissible R5 SHIV that represents a valuable tool to test candidate AIDS vaccines targeting HIV-1 clade C Env.

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.

Postnatal pre- and postexposure passive immunization strategies: protection of neonatal macaques against oral simian-human immunodeficiency virus challenge.

Simian-human immunodeficiency viruses (SHIV) allow the evaluation of antiviral strategies that target the envelope glycoproteins of the human immunodeficiency virus 1 (HIV-1) in macaques. We previously protected neonates from oral challenge with cell-free SHIV-vpu+ by passive immunization with synergistic human neutralizing monoclonal antibodies (mAbs) (Baba et al., Nat Med 6:200-206, 2000). mAbs were administered prenatally to pregnant dams and postnatally to the neonates. Here, we used solely postnatal or postexposure mAb treatment, thus significantly reducing the amount of mAbs necessary. All neonatal monkeys were also protected with these abbreviated mAb regimens. Our results are directly relevant for humans because we used mAbs that target HIV-1 envelope glycoproteins. Thus, the large-scale use of passive immunization with neutralizing mAbs may be feasible in human neonates. The mAbs, being natural human proteins, can be expected to have low toxicity. Passive immunization has promise to prevent intrapartum as well as milk-borne virus transmission from HIV-1-infected women to their infants.

DNA prime/protein boost vaccine strategy in neonatal macaques against simian human immunodeficiency virus.

Newborn macaques were vaccinated against a chimeric simian human immunodeficiency (SHIV) virus, SHIV-vpu+, by DNA priming and boosting with homologous HIV-1 gp160. Following SHIV-vpu+ challenge, containment of infection was observed in 4 of 15 animals given DNA priming/protein boost vaccination and in three of four animals given gp160 boosts only. Rechallenge with homologous virus of six animals that contained the first challenge virus resulted in rapid viral clearance or low viral loads. Upon additional rechallenge with heterologous, pathogenic SHIV89.6P, four of these six animals maintained normal CD4+ T-cell counts with no or limited SHIV89.6P infection. Our data suggest that humoral and cellular immune mechanisms may have contributed to the containment of SHIV89.6P; however, viral interference with SHIV-vpu+ could also have played a role. Our results indicate that immunogenicity and efficacy of candidate AIDS vaccines are not affected when vaccination is initiated during infancy as compared with later in life.

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.

Heterologous neutralizing antibody induction in a simian-human immunodeficiency virus primate model: lack of original antigenic sin.

According to the principle of original antigenic sin, neutralizing antibodies (NAbs) initially directed against a single virus strain compromise the immune system's ability to subsequently mount adequate responses against antigenically divergent virus strains. In this study, rhesus macaques, after vaccination and breakthrough infection with homologous simian-human immunodeficiency virus (SHIV), developed strong SHIV-IIIB strain-directed NAb responses that were mostly V3 loop specific. After superinfection with heterologous SHIV89.6P, all macaques developed high-titer SHIV89.6P-specific NAbs without significant boosting of SHIV-IIIB-specific NAbs. These results indicate that prior B cell responses against a single immunodeficiency virus strain do not preclude the later development of NAbs against a divergent strain of the same virus.

Extensively deleted simian immunodeficiency virus (SIV) DNA in macaques inoculated with supercoiled plasmid DNA encoding full-length SIVmac239.

Using long-distance DNA PCR, we prospectively followed rhesus monkeys that had been inoculated intramuscularly with supercoiled plasmid DNA encoding intact simian immunodeficiency virus (SIV). From 4 to 10 weeks postinoculation onward, we detected extensively deleted proviral genomes along with full-length viral genomes in peripheral blood mononuclear cells (PBMC) in adult macaques. During their chronic asymptomatic phase of infection, the frequency of deleted proviral genomes was similar in PBMC and lymph nodes. The latter, however, harbored significantly more full-length proviral DNA than PBMC, consistent with the lack of effective antiviral cytotoxic T-cell activity in lymph nodes described by others during human immunodeficiency virus infection. After the macaques progressed to AIDS, full-length proviral DNA became equally abundant in lymph nodes and in PBMC. We have demonstrated that although a single molecular species of proviral DNA was inoculated, genomic diversity was detected within a short time, thus confirming the genetic instability of the SIV genome in vivo.

Protection of neonatal macaques against experimental SHIV infection by human neutralizing monoclonal antibodies.

Neonatal macaques were completely protected against oral challenge with SHIV-vpu+, a simian-human immunodeficiency virus that encodes the envelope gene of a laboratory-adapted HIV strain, by pre- and post-natal treatment with a triple combination of human neutralizing monoclonal antibodies (mAbs). The mAbs were directed either against the CD4 binding site, a glycosylation-dependent gp120 epitope, or against a linear epitope on gp41. This triple combination was highly synergistic in vitro and neutralized primary HIV completely. Subsequently, oral challenge was performed with pathogenic SHIV89.6P, an animal-passaged variant of a chimeric virus that encodes the envelope gene of the primary, dual-tropic HIV89.6. Only post-natal treatment with a similar triple mAb combination was used. One out of 4 mAb-treated infants was completely protected from infection. In the other 3 treated animals, there was a tendency towards lower peak viral RNA loads compared with untreated controls. Two out of 4 mAb-treated infants maintained normal CD4+ T-cell numbers, in contrast to all controls that had steep declines at 2 weeks post-challenge. We conclude that the triple mAb combination significantly protected the neonates, even against mucosal challenge with pathogenic SHIV89.6P. Passively administered synergistic human mAbs may play a role in preventing mother-infant transmission of HIV, both against intrapartum transmission as well as against infection through breast milk. As passive immunization is a tool to assess correlates of immune protection, we conclude that the epitopes recognized by the mAbs in our combinations are important for AIDS vaccine development. Future passive immunization studies may reveal other important conserved epitopes.

Passive immunization against oral AIDS virus transmission: an approach to prevent mother-to-infant HIV-1 transmission?

To develop immunoprophylaxis regimens against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, we established a simian-human immunodeficiency virus (SHIV) model in neonatal macaques that mimics intrapartum mucosal virus exposure (T.W. Baba, J. Koch, E.S. Mittler et al: AIDS Res Hum Retroviruses 10:351-357, 1994). We protected four neonates from oral SHIV-vpu+ challenge by ante- and postpartum treatment with a synergistic triple combination of immunoglobulin (Ig) G1 human anti-HIV-1 neutralizing monoclonal antibodies (mAbs) (T.W. Baba, V. Liska, R. Hofmann-Lehmann et al: Nature Med 6:200-206, 2000), which recognize the CD4-binding site of Env, a glycosylation-dependent gp120, or a linear gp41 epitope. Two neonates that received only postpartum mAbs were also protected from oral SHIV-vpu+ challenge, indicating that postpartum treatment alone is sufficient. Next, we evaluated a similar mAb combination against SHIV89.6P, which encodes env of primary HIV89.6. One of four mAb-treated neonates was protected from infection and two maintained normal CD4+ T-cell counts. We conclude that the epitopes recognized by the three mAbs are important determinants for achieving protection. Combination immunoprophylaxis with synergistic mAbs seems promising to prevent maternal HIV-1 transmission in humans.

Potent neutralization of primary human immunodeficiency virus clade C isolates with a synergistic combination of human monoclonal antibodies raised against clade B.

OBJECTIVES: We investigated the ability of several human neutralizing monoclonal antibodies (mAbs), originally raised against human immunodeficiency virus (HIV) clade B isolates, to neutralize primary clade C isolates as single agents and in combination. STUDY DESIGN/METHODS: HIV clade C isolates from five different countries were tested for susceptibility to neutralization by anti-clade B mAbs in human peripheral blood mononuclear cells. Monoclonal antibody combinations were evaluated for possible synergy. RESULTS: All 20 primary HIV clade C isolates could be neutralized 97.5% to 100% by a quadruple combination of mAbs IgG1b12, 2G12, 2F5, and 4E10. These mAbs recognized conserved epitopes and were highly synergistic, resulting in strong cross-clade neutralization. CONCLUSIONS: In our previous experiment, a synergistic combination of human neutralizing mAbs protected all macaque neonates against oral challenge with a simian-human immunodeficiency virus encoding HIV env. Together, our data suggest that passive immunization with currently available anti-clade B mAbs could play a role in preventing HIV clade C transmission through breastfeeding.

Postnatal passive immunization of neonatal macaques with a triple combination of human monoclonal antibodies against oral simian-human immunodeficiency virus challenge.

To develop prophylaxis against mother-to-child human immunodeficiency virus (HIV) transmission, we established a simian-human immunodeficiency virus (SHIV) infection model in neonatal macaques that mimics intrapartum mucosal virus exposure (T. W. Baba et al., AIDS Res. Hum. Retroviruses 10:351-357, 1994). Using this model, neonates were protected from mucosal SHIV-vpu(+) challenge by pre- and postnatal treatment with a combination of three human neutralizing monoclonal antibodies (MAbs), F105, 2G12, and 2F5 (Baba et al., Nat. Med. 6:200-206, 2000). In the present study, we used this MAb combination only postnatally, thereby significantly reducing the quantity of antibodies necessary and rendering their potential use in humans more practical. We protected two neonates with this regimen against oral SHIV-vpu(+) challenge, while four untreated control animals became persistently infected. Thus, synergistic MAbs protect when used as immunoprophylaxis without the prenatal dose. We then determined in vitro the optimal MAb combination against the more pathogenic SHIV89.6P, a chimeric virus encoding env of the primary HIV89.6. Remarkably, the most potent combination included IgG1b12, which alone does not neutralize SHIV89.6P. We administered the combination of MAbs IgG1b12, 2F5, and 2G12 postnatally to four neonates. One of the four infants remained uninfected after oral challenge with SHIV89.6P, and two infants had no or a delayed CD4(+) T-cell decline. In contrast, all control animals had dramatic drops in their CD4(+) T cells by 2 weeks postexposure. We conclude that our triple MAb combination partially protected against mucosal challenge with the highly pathogenic SHIV89.6P. Thus, combination immunoprophylaxis with passively administered synergistic human MAbs may play a role in the clinical prevention of mother-to-infant transmission of HIV type 1.

Sensitive and robust one-tube real-time reverse transcriptase-polymerase chain reaction to quantify SIV RNA load: comparison of one- versus two-enzyme systems.

Plasma viral RNA load is a key parameter in disease progression of lentiviral infections. To measure simian immunodeficiency virus (SIV) RNA loads, we have established a quantitative one-tube assay based on TaqMan chemistry. This real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has advantages compared with previous methods, such as higher sensitivity, shorter time consumption, and low risk of cross-contamination. The sensitivity of the assay was optimized by comparing different enzyme systems. The one-enzyme protocol using rTth DNA polymerase was superior to two assays employing two enzymes. It detects 100% of the samples containing four copies of RNA transcript and allows quantification of viral RNA loads over an 8-log unit dynamic range. As few as 50 copies per milliliter of plasma can be detected within RNA extracted from 140 microl of plasma. This is especially relevant in studies employing neonatal macaques, from which only small volumes of blood can be sampled, and in studies in which low viral RNA loads are expected. Because of the use of rTth DNA polymerase, DNA contamination can be avoided by carryover prevention with uracil N-glycosylase (UNG). We demonstrate that for optimization of real-time PCR sensitivity, not only concentrations of different reagents but also different enzyme systems must be evaluated. Our assay facilitates and enhances the quantification of plasma RNA loads, a critical parameter for many studies, including evaluations of vaccine candidates or antiviral regimens.

1999: a time to re-evaluate AIDS vaccine strategies.

The field of AIDS vaccine development is in flux. Important new findings were reported in 1999 that led to a rethinking of AIDS vaccine strategies. We have been given the challenging task of providing an overview. Rather than attempting to provide a comprehensive summary, we will restrict our discussion to a few major topics, and we ask for understanding if we can only highlight.

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.

Live attenuated AIDS viruses as vaccines: promise or peril?

Live attenuated viruses can provide vaccine protection against various viral illnesses. A number of live attenuated strains of the simian immunodeficiency virus (SIV) or related lentiviruses have been evaluated in primate models as vaccine candidates against AIDS. Impressive efficacy was observed for some viruses, most notably SIV strains with deletions in the nef-gene. Sterilizing immunity was seen against homologous and heterologous virus challenge, against cell-free and cell-associated challenge, against intravenous and mucosal challenge, and against challenge as early as 3 weeks and as late as 2.25 years after just one immunization. However, these promising efficacy results are overshadowed by safety problems, such as reversion of the vaccine strain to a pathogenic virus encoding full-length nef or residual virulence of multiply deleted vaccine strains. Strategies aimed at decreasing the replicative capacity of nef-deleted vaccine strains to increase the safety profile have significantly curtailed vaccine efficacy. Nevertheless, studies of live attenuated vaccine strains should proceed and should focus on determining the correlates of vaccine protection and the molecular determinants for virulence and attenuation.

Persistent infection of rhesus macaques by the rev-independent Nef(-) simian immunodeficiency virus SIVmac239: replication kinetics and genomic stability.

We generated previously a Nef(-), replication-competent clone of SIVmac239 in which the Rev protein and the Rev-responsive element were replaced by the constitutive transport element (CTE) of simian retrovirus type 1 (A. S. von Gegerfelt and B. K. Felber, Virology 232:291-299, 1997). In the present report, we show that this virus was able to infect and replicate in rhesus macaques. The Rev-independent Nef(-) simian immunodeficiency virus induced a persistent humoral immune response in all monkeys, although viral loads were very low. Upon propagation in the monkeys, the genotype remained stable and the virus retained its in vitro growth characteristics. The infected monkeys showed normal hematological values and no signs of disease at more than 18 months post-virus exposure. Therefore, replacement of the essential Rev regulation by the CTE generated a virus variant that retained its replicative capacity both in vitro and in vivo, albeit at low levels.

Viremia and AIDS in rhesus macaques after intramuscular inoculation of plasmid DNA encoding full-length SIVmac239.

We have succeeded in stably maintaining the entire genome of SIVmac239 as a plasmid clone. Supercoiled proviral plasmid DNA was inoculated intramuscularly into two adult rhesus macaques and into a neonate. All three animals became viremic and seroconverted. Viral kinetics were followed prospectively by quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR), measurement of proviral DNA load in peripheral blood mononuclear cells (PBMCs) by PCR, and virus isolation by cocultivation. The infant developed high virus loads and succumbed to AIDS and SIV-associated nephropathy at 10 weeks postinoculation. Both adults are still living but have progressed to AIDS; one adult has also developed severe thrombocytopenia. We conclude that infection through intramuscular inoculation of cloned plasmid DNA encoding the entire proviral genome is reproducible and will provide a useful tool for studying viral pathogenesis.

Oral transmission of primate lentiviruses.

Oral transmission of human immunodeficiency virus type 1 (HIV-1) is well documented in children who become infected postnatally through breast milk. In contrast, epidemiologic surveys have yielded conflicting data regarding oral HIV-1 transmission among adults, even though case reports have described seroconversion and the development of AIDS in adults whose only risk was oral-genital contact. To study oral virus transmission in primate models, we exposed rhesus macaques of various ages to cell-free simian immunodeficiency virus (SIV), including uncloned and molecularly cloned viruses. In neonates, viremia and AIDS developed after nontraumatic oral exposure to several SIV strains. Furthermore, chimeric simian human immunodeficiency viruses containing the HIV-1 envelope can also cross intact upper gastrointestinal mucosal surfaces in neonates. In adult macaques, infection and AIDS have resulted from well-controlled, nontraumatic, experimental oral exposure to different strains of SIV. These findings have implications for the risks of HIV-1 transmission during oral-genital contact.