ESG and LM designed the study. ESG and LM analyzed the data. TM and GG enrolled patients. DM provided reagents and technical advice. ESG, TM, GG, DM, and LM contributed to writing the paper. ESG collected data and performed experiments for the study.
The authors have declared that no competing interests exist.
A Phase I clinical trial has been proposed that uses neutralising monoclonal antibodies (MAbs) as passive immunoprophylaxis to prevent mother-to-child transmission of HIV-1 in South Africa. To assess the suitability of such an approach, we determined the sensitivity of paediatric HIV-1 subtype C viruses to the broadly neutralising MAbs IgG1b12, 2G12, 2F5, and 4E10.
The gp160 envelope genes from seven children with HIV-1 subtype C infection were cloned and used to construct Env-pseudotyped viruses that were tested in a single-cycle neutralisation assay. The epitopes defining three of these MAbs were determined from sequence analysis of the envelope genes. None of the seven HIV-1 subtype C pseudovirions was sensitive to 2G12 or 2F5, which correlated with the absence of crucial N-linked glycans that define the 2G12 epitope and substitutions of residues integral to the 2F5 epitope. Four viruses were sensitive to IgG1b12, and all seven viruses were sensitive to 4E10.
Only 4E10 showed significant activity against HIV-1 subtype C isolates, while 2G12 and 2F5 MAbs were ineffective and IgG1b12 was partly effective. It is therefore recommended that 2G12 and 2F5 MAbs not be used for passive immunization experiments in southern Africa and other regions where HIV-1 subtype C viruses predominate.
AIDS is caused by HIV. By killing the cells of the body's immune system, HIV infection makes people vulnerable to many potentially fatal bacterial and viral diseases. HIV is most commonly spread through unprotected sex with an infected partner but it can also pass from mother to child during late pregnancy or birth, or through breast milk. At least one in four infected women will transmit HIV to their babies if left untreated. But if infected women are treated with drugs that fight HIV—so-called antiretrovirals—during late pregnancy and if breastfeeding does not occur, only one to two babies in 100 will become infected with HIV. In addition, elective Caesarian section has been found to be protective against HIV infection. Implementation of this approach has greatly reduced mother-to-child transmission in developed countries, but most HIV-infected women live in developing countries where access to antiretrovirals is limited. In these cases, treatment of pregnant women (during pregnancy and delivery) and their newborn babies with a single dose of one antiretroviral drug, which can halve HIV transmission, is used, even though WHO/UNAIDS recommends simple antenatal, intrapartum, and postnatal antiretroviral regimens to achieve levels of less than 5% transmission in resource poor settings. These strategies will not have an impact on breastmilk transmission, which accounts for half the transmissions in these settings.
One way to reduce breastmilk transmission of HIV might be by “passive immunization.” In this, newborn babies would be injected with HIV-specific antibodies—proteins that stick to molecules on the surface of HIV. Because the virus uses these molecules to invade the baby's immune cells, injected antibodies might stop HIV from the mother becoming established in her offspring. Four antibodies have been made in the laboratory—so-called human monoclonal antibodies—that bind to the surface of HIV subtype B, which is found mainly in Europe and North America, and stop HIV from killing human cells. However, most HIV isolated in Africa is subtype C, so in this study researchers have tested whether these antibodies prevent HIV subtype C killing cells grown in the laboratory. It is important, they argue, that antibodies should be shown to work outside the body before testing passive immunization in babies.
The researchers isolated several subtype C viruses from babies born in Johannesburg, South Africa, and made artificial viruses (known as “pseudotyped” viruses) from them. These artificial viruses could then be used in tests to see whether the human monoclonal antibodies could prevent the viruses infecting human cells in a laboratory test, that is, whether the viruses were “sensitive” to the antibodies. All the viruses were insensitive to two of the antibodies (2G12 and 2F5), and the researchers show that this was because the viruses lacked the specific parts of the HIV surface molecules recognized by these antibodies. Four of the viruses were sensitive to an antibody called IgG1b12, and all were sensitive to antibody 4E10, albeit at high concentrations that might be difficult to achieve in people. Finally, the researchers report that the sensitivity of the viruses was not enhanced by using all four antibodies at the same time.
Given these results, the researchers warn against using 2G12 and 2F5 antibodies for passive immunization to prevent mother-to-child transmission, in particular postnatal transmission, in areas where most people are infected with HIV subtype C viruses. Furthermore, because animal studies have indicated that only combinations of at least three monoclonal antibodies with activity against HIV in laboratory tests provide complete protection against HIV infection, the researchers question whether any clinical trials on passive immunization should be started with currently available antibodies. Their doubts about such trials are heightened by observations that 4E10 and 2F5 react against antigens present on human cells, which might make them unsafe for use in people, although so far no adverse effects have been seen in adults treated with these antibodies. However, these experiments used an artificial laboratory-based assay and it's possible that these antibodies might kill HIV subtype C more effectively in people; other components of the immune system might help them deal with the virus. If clinical studies of these antibodies do go ahead, it is essential that the babies in these trials must be carefully monitored to ensure that the antibodies are safe, and they and their mothers should also be given access to optimal antiretroviral prophylaxis according to WHO/UNAIDS guidelines. In a related
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Assessment of viruses from seven children with HIV-1 subtype C infection showed generally poor sensitivity to four monoclonal antibodies proposed for a trial of passive immunoprophylaxis to prevent mother-to-child transmission.
Only four broadly neutralising monoclonal antibodies (MAbs) against HIV-1 have been generated to date, all of which were derived from patients with HIV-1 subtype B infection. IgG1b12 recognizes an epitope overlapping the CD4 binding site in the envelope glycoprotein complex [
Mother-to-child transmission (MTCT) of HIV-1 infection remains a significant problem in developing countries. While the use of single-dose nevirapine, acting to prevent intrapartum transmission, has reduced the number of infections, more potent interventions are needed, particularly to prevent postpartum transmissions. It is estimated that in South Africa alone, approximately 96,000 children with HIV-1 infection were born in 2003 [
The most common subtype of HIV-1 infection in southern Africa as well as globally is subtype C (
Viruses were isolated from the blood of children with HIV-1 infection by standard co-culture techniques using peripheral blood mononuclear cells (PBMCs) [
Patient Information and Viral Isolate Characteristics for HIV-1 Subtype C Cloned Envelope Genes
MAbs were obtained from the National Institutes of Health Reference and Reagent Program (Germantown, Maryland, United States) and the International AIDS Vaccine Initiative Neutralizing Antibody Consortium (New York, New York, United States), and used at a starting concentration of 50 μg/ml. Recombinant soluble CD4 (sCD4) comprising the extracellular domain of human CD4 produced in Chinese hamster ovary cells was obtained from Progenics Pharmaceuticals (Tarrytown, New York, United States), and tested at 50 μg/ml. Two plasma samples (BB12 and IBU21) from blood donors with HIV-1 subtype C infection were tested at a starting dilution of 1:50.
JC53-bl cells were obtained from the National Institutes of Health Reference and Reagent Program (catalog number 8129). These cells were derived from a HeLa cell clone that expresses CD4, CCR5, and CXCR4 constitutively [
Proviral DNA extracted from in vitro infected PBMCs was used to amplify full-length envelope genes. The 3-kilobase PCR fragments, generated using envA and envM primers [
Neutralisation was measured as a reduction in luciferase gene expression after a single-round infection of JC53-bl cells with Env-pseudotyped viruses [
Cloned
We cloned complete (gp160) envelope genes from seven HIV-1 subtype C isolates cultured from the blood of children with perinatally acquired HIV-1 infection. Five of these isolates were from rapidly progressing infants (RP and COT) who developed severe clinical symptoms within the first year of life, most of whom died shortly after blood collection (
The HIV-1 subtype C envelope clones were used to generate Env-pseudotyped viruses by co-transfection with a subgenomic plasmid. These pseudoviruses were tested for their sensitivity to neutralisation by the MAbs IgG1b12, 2G12, 2F5, and 4E10. The MAbs 2G12 and 2F5 failed to neutralise any of the seven HIV-1 subtype C pseudoviruses at 50 μg/ml, whereas the HIV-1 subtype B virus QH692.42 had IC50 values of 0.8 and 7.1, respectively (
Sensitivity of HIV-1 Subtype C Pseudovirions to Anti-HIV MAbs, sCD4, and Plasma
Synergistic neutralisation among MAbs that recognize different specificities in the envelope glycoprotein has been suggested [
Analysis of the dose-response curves confirmed the lack of significant synergy among MAbs. Those viruses sensitive to IgG1b12 (RP4.3, RP6.6, TM7.9, and COT9.6) had similar neutralisation curves in the presence of IgG1b12 alone or when tested as part of TriMAb with or without 4E10 (
The MAb concentrations in the triple and quadruple combination are represented as the concentration of each MAb in the equimolar mix starting at 50 μg/ml. Results are shown as the reduction of virus infectivity relative to the virus control (without MAbs) with 50% inhibition indicated by a dotted line. Note those viruses sensitive to IgG1b12 and 4E10 (A) and those viruses sensitive to 4E10 alone (B).
Given the relative resistance of the HIV-1 subtype C pseudovirions to neutralisation by MAbs, we chose to test their responses to sCD4 and polyclonal antibodies from individuals with HIV-1 infection. sCD4, which blocks gp120 binding to the CD4 receptor, neutralised all of the pseudovirions (
All pseudovirions except TM7.9 were neutralised by one or both of the plasma samples with a wide variation in IC50 titres, as is often seen when using polyclonal antibodies, suggesting that these envelopes were not atypical in their ability to be neutralised (
Sequence analysis of the predicted N-linked glycosylation (PNG) sites at positions 295, 332, and 392, which are critical for the 2G12 epitope, indicated that all HIV-1 subtype C isolates lacked the glycan 295. TM7.9 also lacked the glycan 392 (
Amino Acid Sequences of MAb Epitopes in Cloned Subtype C Envelope Genes
The 2F5 epitope is centred on the sequence ELDKWA [
4E10 recognizes an epitope containing the sequence NWF(D/N)IT [
The neutralisation sensitivity of HIV-1 subtype C isolates derived from children appears similar to previously reported sensitivity of isolates from adults with HIV-1 subtype C infection [
In this study, we have used cloned envelope genes in a single-cycle neutralisation assay, which is a high-throughput assay that, to our knowledge, is rapidly becoming the method of choice for measuring antibody neutralisation [
It has been shown in multiple studies that 2G12 is generally ineffective against HIV-1 subtype C isolates [
The 2F5 MAb has been shown to have broadly neutralising activity but has minimal efficacy against HIV-1 subtype C viruses [
Our data with IgG1b12 agree with other studies in that this MAb is more effective than 2F5 or 2G12 at neutralising HIV-1 subtype C viruses, although IgG1b12 inhibited only approximately 50% of the isolates tested [
The 4E10 epitope appears to be the most broadly cross-reactive MAb described to date, neutralising all viruses so far tested. In previous studies, 4E10 has been shown to neutralise 100% of viruses in a comprehensive panel that included all genetic subtypes of HIV-1 group M and some recombinant forms [
Some studies have suggested that MAbs can act synergistically to increase neutralisation potency against HIV-1 [
The MTCT of HIV-1 infection is usually associated with transmission of single variants [
Based on our results, we question the use of MAb combinations that include 2F5 and 2G12 as a prophylactic treatment in regions where HIV-1 subtype C viruses predominate, even if such combinations were to include 4E10 and IgG1b12. In passive immunoprophylaxis studies using a single MAb, protection was not observed even when the challenge strain was successfully neutralised in vitro. Only a combination of at least three MAbs with bona fide neutralisation activity against the challenge strain offered complete protection [
Overall, we believe that the use of these MAbs to prevent MTCT of HIV-1 subtype C infection is unlikely to be efficacious; therefore, a clinical trial should not be conducted. A recent study has confirmed our viewpoint that these MAbs would have limited benefit when used to prevent MTCT in populations with HIV-1 non-B subtype infection [
The study of the epitopes recognized by these broadly neutralising MAbs contributes to the knowledge necessary for the rational design of an immunogen capable of inducing a broad and potent neutralisation response against HIV-1 infection. Considerable efforts have been invested in designing immunogens based on these epitopes [
The GenBank (
We thank Dennis Burton, James Binley, and the National Institutes of Health Reference and Reagent Program for supplying MAbs and Progenics for supplying sCD4. We are grateful to Penny Moore for her help with the sequence analysis and the critical reading of the manuscript.
inhibitor concentration
monoclonal antibody
mother-to-child transmission
peripheral blood mononuclear cell
predicted N-linked glycosylation
soluble CD4
tissue culture infectious dose