Vaccines to prevent transmission of HIV-1 via breastmilk: scientific and logistical priorities

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Vaccines to prevent transmission of HIV-1 via breastmilk: scientific and logistical priorities Katherine Luzuriaga, Marie-Louise Newell, Francois Dabis, Jean-Louis Excler, John L Sullivan

Mother-to-child transmission (MTCT) of HIV-1 is the major mode of paediatric infection. The rapidly increasing incidence of MTCT worldwide has resulted in an urgent need for preventive strategies. Antiretroviral regimens can prevent intrapartum HIV transmission; however, these regimens do not prevent HIV transmission through breastfeeding. Furthermore, children who escape MTCT are again at risk of infection when they become sexually active as adolescents. An infant vaccine regimen, begun at birth, would hence be a more attractive strategy and might also provide the basis for lifetime protection. Unique features of MTCT and paediatric HIV disease could be helpful in understanding correlates of immune protection and could facilitate rapid assessment of vaccine efficacy. Thus, there is compelling rationale to develop safe, effective HIV vaccines for use in infants and children. Here, we discuss the scientific and logistical challenges for the development of paediatric HIV vaccines; available vaccines and completed or planned paediatric vaccine trials are also discussed. A preventive vaccine is the best long-term strategy to control the HIV-1 pandemic. The Global HIV Vaccine Enterprise1 has outlined a scientific strategic plan for HIV vaccine development.2 Key features of this plan include defining fundamental scientific barriers to the development of an effective preventive vaccine, creating vaccine centres charged with advancing the rational design and development of vaccines, and establishing standard preclinical and clinical laboratory assessments of candidate vaccines. To date, most efforts have focused on the development of vaccines for adults. However, infants, children, and adolescents constitute major risk groups for HIV infection, and morbidity and mortality are particularly high in infants infected with the virus. An effective neonatal vaccine could prevent transmission of the virus via breastmilk and provide the basis for lifetime immunity. Here, we discuss the rationale for the development of HIV vaccines for infants and outline important scientific and logistical considerations for their development.

Rationale for paediatric HIV vaccines Rising incidence and prevalence of infection Since the beginning of the HIV pandemic 25 years ago, an estimated 60 million individuals have been infected with the virus; 39 million now live with the infection and almost 5 million acquire the virus every year.3 HIV infection disproportionately affects women and young people, with half of all new infections arising in individuals aged 15–24 years. Heterosexual transmission remains the predominant mode of transmission worldwide, and about half of all newly infected individuals are female, particularly those in their early to middle teenage years.3 The rising incidence and prevalence of HIV in women of childbearing age have greatly increased the number of children infected. In 2003, an estimated 2·2 million children had HIV. About 2 million children are born to women infected with HIV every year, and every one of them is at risk of infection; 740 000 children younger than age www.thelancet.com Vol 368 August 5, 2006

15 years were newly infected in 2004, most through mother-to-child transmission (MTCT). This figure represents almost 2000 new infections per day (15% of all new infections). More than 90% of paediatric infections occur in sub-Saharan Africa, where their increase has reversed advances in child mortality achieved through immunisation and public-health programmes over the latter decades of the 20th century.

Limitations of antiretroviral efficacy in reducing MTCT

Lancet 2006; 368: 511–21 University of Massachusetts Medical School, Program in Molecular Medicine, 373 Plantation Street, Suite 318, Worcester, MA 01605, USA (Prof K Luzuriaga MD, Prof J L Sullivan MD); Centre for Paediatric Epidemiology, Institute of Child Health, London, UK (Prof M-L Newell PhD); INSERM U593, Institut de Santé Publique, Epidémiologie et Dévelopment (ISPED), Université Victor Segalen, Bordeaux, France (Prof F Dabis MD); and International AIDS Vaccine Initiative, New Delhi, India (J-L Excler MD) Correspondence to: Dr Katherine Luzuriaga katherine.luzuriaga@ umassmed.edu

Maternal or perinatal antiretroviral prophylaxis regimens can greatly reduce MTCT of HIV.4 In a landmark study (Paediatric AIDS Clinical Trial Group 076),5 treatment of American and European pregnant women and their infants with zidovudine resulted in a transmission rate of 8% compared with a transmission rate of 26% in the placebo group (67% reduction in MTCT). By use of a combination of antiretroviral therapies, overall MTCT rates have dropped to below 2% in most of the developed world.6,7 Similarly, the shortest possible antiretroviral regimen, consisting of a single dose of nevirapine administered to a woman during delivery followed by a single dose of nevirapine administered to the baby shortly after birth, greatly reduced the risk of intrapartum HIV transmission in Africa.8 However, even this regimen has been difficult to implement widely in resource-poor settings; despite an offer of corporate donation of nevirapine to prevention of MTCT (PMTCT) programmes, many women who

Search strategy and selection criteria We identified data for this review by searching PubMed for relevant articles published in English until November, 2005, using the following terms: “HIV vaccines“, alone or in combination with “child“; and “modified vaccinia Ankara“, “poxvirus vaccines“, or “DNA vaccines“ in combination with “HIV“. We also identified numerous articles through searches of our personal files.

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could benefit from its use probably do not receive the drug because the necessary infrastructure and manpower to deliver the intervention are lacking. Furthermore, findings of studies9–11 indicate the rapid selection of viruses with resistance mutations after the use of one or two antiretroviral drugs in the setting of incomplete control of maternal viral replication. These data raise concerns that the usefulness of the drug most commonly available in resource-limited settings to prevent MTCT might be reduced with more widespread use. Perinatal antiretroviral regimens do not prevent transmission of HIV through breastfeeding, although randomised trials12 of other regimens that might are underway. A vaccine regimen started at birth would not only prevent MTCT, but might also provide the basis for lifetime protection against HIV infection. Use of existing global infrastructures for the delivery of routine childhood vaccines would enhance the feasibility of this approach.

Assessment of vaccine efficacy Findings of clinical trials indicating antiretroviral treatment efficacy in PMTCT provided proof-of-concept regarding the feasibility of preventing HIV transmission between individuals.13 Trials of HIV vaccines in breastfeeding infants might similarly yield evidence of vaccine efficacy or correlates of immune protection more rapidly than vaccine trials in adults. Natural history studies of MTCT and interventional trials of PMTCT have organised appropriate cohorts and defined transmission and incidence rates. At-risk mother-infant pairs can be easily identified through voluntary counselling and testing in antenatal clinics; many centres have proven their ability to enrol and follow up mothers and their infants in PMTCT trials. Within these research settings, an infant can be diagnosed with HIV within days or weeks of infection. Finally, if a vaccine does not provide sterilising immunity, the high, early viral loads and rapid tempo of disease progression in infants14–16 could allow rapid assessment of vaccine efficacy in lowering viral load or altering disease outcome after infection. Concurrent assessment of vaccine-induced immune responses could lead to the identification of potential correlates of immune protection. In this respect, a parallel can be drawn with data from a randomised controlled trial17 in Mozambique of a malaria protein subunit vaccine (RTS, S/ASO2A, GlaxoSmithKline Biologicals, Rixensart, Belgium), which showed a 30% reduction in clinical episodes and a 58% reduction in episodes of severe malaria in a cohort of vaccinated children aged 1–5 years. There is, therefore, compelling rationale for the rapid development of safe, effective HIV vaccines for use in newborn children. But what are the scientific and operational research issues associated with their development? 512

Pathogenesis of MTCT and vaccine development Transmission of HIV via breastmilk In the absence of antiretroviral therapy, 25–30% of women infected with HIV transmit the virus to their infants. MTCT can occur during gestation, during delivery, or postpartum through breastfeeding. In nonbreastfed populations, 25–30% of infected infants have detectable provirus in their peripheral blood lymphocytes at birth, suggesting that they were infected in utero.18 In the remaining 70–75%, HIV RNA or provirus is not detected in the peripheral blood at birth, but becomes detectable within weeks of birth, compatible with the intrapartum transmission of HIV. In breastfed populations, breastfeeding probably accounts for a third to half of all infections.19,20 In many areas—eg, the USA, Europe, Brazil, and Thailand—the availability, affordability, safety, and cultural acceptability of formula feeding have contributed to the feasibility of using formula to prevent HIV transmission through breastmilk. However, in resourcelimited settings, breastfeeding remains crucial for infant health and survival.21,22 In the regions with the highest HIV incidence and prevalence, the high cost of formula, poor hygienic conditions, and lack of potable water and refrigeration make formula feeding impractical and potentially dangerous. Although data from some studies19,23 suggest that transmission via breastmilk commonly occurs within the first months of life, findings of a recent meta-analysis20 indicate a constant risk of transmission (8·9 transmissions per 100 child-years of breastfeeding) between age 1 month and 18 months. The probability of transmission has been estimated at 0·00064 per L ingested and 0·00028 per day of breastfeeding; the latter is roughly equivalent to the probability of HIV transmission per unprotected sex act between adults.24 Low CD4-cell counts and high plasma HIV RNA copy numbers are associated with the highest risk of breastmilk transmission.20,25 Rousseau and colleagues26 have shown that every log10 increase in breastmilk cell-associated virus is associated with a three-fold increase in the risk of MTCT. However, oral transmission of cell-free simian immunodeficiency virus (SIV) has also been documented in infant macaques.27

Viral pathogenesis of early paediatric infection Clinical manifestations of HIV infection in children differ greatly from those in adults. Studies from the USA, Europe,28,29 and Africa30 have documented faster rates of disease progression and death in infants than in adults. HIV-associated morbidity and mortality seem to be particularly high in African cohorts; results of a pooled analysis30 revealed that about a third of children with HIV infection die by their first birthday and more than half by the age of 2 years, with children infected in utero or intrapartum having the fastest progression. www.thelancet.com Vol 368 August 5, 2006

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Although the precise mechanisms of MTCT are unknown, intrapartum transmission probably results from a discrete exposure to virus in maternal blood or cervicovaginal secretions across infant mucosal surfaces, whereas breastmilk transmission is thought to result from many low-dose exposures across the gastrointestinal tract. The relative contribution of cell-free versus cellassociated viruses in intrapartum and breastmilk transmission remains unclear. Models of transmission suggest that HIV first infects submucosal macrophages or dendritic cells and that these cells then transmit the virus to CD4-positive T cells. These cells constitute the short-lived, productively infected population of cells that produce the majority of plasma virus in the absence of therapy31,32 and might constitute an important latent viral reservoir in individuals on suppressive antiretroviral therapy.33,34 Viruses isolated in early vertical infection most often use the CCR5 co-receptor, although occasional use of other receptors has been reported. Over the first few weeks of vertical infection, plasma HIV RNA copy numbers commonly exceed 10⁵–10⁷ per mL.15,16,35,36 Peak and set-point plasma HIV RNA concentrations are significantly higher in antiretroviral-naive Kenyan infants than in adults. In one study,14 the average plasma HIV RNA set point in infants who acquired HIV infection in utero or before age 2 months was almost 1 log higher than in infants who acquired the infection through breastfeeding after 2 months of age. Most studies have shown that plasma HIV RNA concentrations remain high in infants over the first 1–2 years of life.15,16 Gradual reductions in plasma HIV RNA copy numbers have been noted thereafter in vertically infected children up to age 5–6 years.37,38 Reductions in plasma HIV RNA after the initiation of antiretroviral treatment are associated with clinical benefit.39,40 Collectively, these data suggest that viral replication is an important determinant of paediatric disease and that the long-term increase in plasma viral load noted during the first 2 years of life probably contributes to the faster disease progression seen in children than in adults. Many factors could contribute to the early, long-term increase in plasma HIV RNA concentrations, including the kinetics of viral replication, the size of the host cell pool permissive to viral replication, and delayed or ineffective virus-specific immune responses. Although relative lymphocytosis and an increased CD4-cell pool size are present throughout infancy and early childhood, the kinetics of viral replication in infants appear to be very similar to those described in adults,32 suggesting that infants might have reduced effector mechanisms to control viral replication.

HIV-specific immune responses in infants Differences in the generation of innate or adaptive immune responses are thought to account for age-related differences in outcome after various viral infections. www.thelancet.com Vol 368 August 5, 2006

Defective, antibody-dependent, cell-mediated cytotoxicity (ADCC) of HIV-infected cells by neonatal natural killer cells has been documented;41 however, this defect appears to reverse by 1 month of age. After the decline in concentrations of passively acquired maternal ADCC antibodies, the active generation of HIV-envelope-specific cytotoxic antibodies by infected infants is delayed.42 The inefficient production of such antibodies to the highly glycosylated envelope protein could indicate the relative inefficiency of antibody production to T-independent antigens in children younger than age 2 years. Protein conjugation has been used successfully to convert T-independent antigens to T-dependent antigens to elicit antibodies to Streptococcus pneumoniae and Haemophilus influenzae in infants.43 The development of HIV-specific binding antibodies in children positive for HIV is well documented; there are fewer studies characterising the development of neutralising antibodies (particularly to autologous viral strains). Reports44,45 of reduced antibody quality and titres in infants immunised against or infected with viruses other than HIV before age 6 months have raised concerns about such infants ability to generate protective antibody responses. However, adult and infant rotavirus-specific B cells have similar variable gene repertoires, suggesting that additional doses of antigen and improved antigen or adjuvant quality could improve the generation of virusspecific antibody responses in infants.46 The fact that the administration of hepatitis B vaccine between birth and 6 months of age is more than 90% effective in preventing MTCT of hepatitis B virus indicates that young infants are capable of making antibodies protective against viral challenge in vivo. Less robust HIV-specific cellular immunity might also preclude the effective containment of viral replication in early infection. Although HIV-specific CD8-positive T-cell responses have been detected in infected infants within the first months of life, they are of lower frequency and are less broad than in adults with primary infection.47–50 Since infants share at least three HLA class I alleles with their mothers, MTCT of CD8-cell escape variants could compromise the generation of early infant CD8-cell responses restricted by shared HLA alleles.51–53 HIV-specific CD8-positive T-cell responses increase in frequency and breadth over the first year of life.49,54 Although the detection of HIV-specific CD8-positive T cells seems to be related to a reduction in viral load during primary infection in adults,55 early HIV-specific CD8-cell responses in infants do not correlate with a reduction in viral load over the first year of life, raising the question of whether the measurement of interferon γ secretion ex vivo accurately indicates CD8-positive T-cell activity in vivo. We53 and others52 have noted that some early CD8-cell responses in infants are associated with the selection of CD8-positive T-cell escape variants, suggesting that CD8-positive T cells can exert selective pressures in vivo. Further characterisation of the ex vivo functional properties of infants’ HIV-specific 513

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As outlined above, many questions remain about the mechanisms of transmission of HIV in breastmilk, early events in neonatal HIV infection, and correlates of immune protection. The neonatal macaque model has been and will probably continue to be helpful in addressing these questions. For example, several studies56 in infant macaques have provided data that lend support to the idea of a protective role of antibodies against HIV transmission. In particular, a combination of monoclonal antibodies administered intramuscularly provided postexposure prophylaxis against intravenous or mucosal exposure to chimeric SIV-HIV in neonatal macaques.57 Although there are concerns that the SIV-HIV challenge viruses used in these studies are highly neutralisation sensitive and might not represent transmitted viruses, these studies have provided compelling data in support of clinical trials of monoclonal antibodies in human infants. One particular infant macaque model,58,59 in which several low doses of uncloned SIV are delivered in breastmilk, seems to be especially promising in its simulation of breastmilk transmission; further studies with this model could provide useful insights into the mechanisms of viral transmission, early events after transmission, potential correlates of protection, and vaccine efficacy.

were detected by ELISA at 24 weeks in 87% of infants immunised with the Chiron vaccine and were significantly higher in vaccine recipients than in infants who received placebo.62 Furthermore, similarly high concentrations of HIV-envelope-specific antibodies were detected by 12 weeks of age in 63% of infants who received an accelerated vaccination schedule (consisting of vaccine administration at birth and at 2, 8, and 20 weeks), suggesting that the vaccine elicited antibody responses despite the presence of high titres of maternal antibodies. Lymphoproliferative responses to HIV proteins were detected more frequently in vaccine recipients (56%) than in controls (14%).63 HIV-specific lymphoproliferative responses were detected by 4 weeks of age in all of 11 infants who received an accelerated vaccine schedule. The results of these phase I trials of recombinant protein vaccines indicate that young infants can generate antibody and lymphoproliferative responses to vaccination even in the presence of high titres of maternal HIV envelope-specific antibodies and when immunised with an accelerated vaccine schedule that would be necessary to protect against MTCT. Neutralising-antibody data have not been reported from these neonatal studies. However, data from adult trials, showing that antibodies induced by subunit vaccines do not neutralise primary HIV isolates, reduced overall enthusiasm for these vaccines and further trials of subunit vaccines alone have not been done in infants. Indeed, the results of two trials64,65 in adults of subunit vaccines indicate no protective effect.

Paediatric vaccine pipeline

Replication-defective recombinant viral vaccines

The induction of long-lasting, antiviral immunity has been achieved most successfully through the use of live attenuated vaccines, probably because these vaccines can emulate the natural infection process, resulting in protective innate and adaptive (humoral and cell-mediated) immunity. Early efforts to develop a live attenuated HIV vaccine were overshadowed by major safety concerns, including the possibility of reversion to virulence by mutation or recombination with superinfecting viruses and chromosomal integration with the attendant risk of malignant transformation.60,61 Efforts to develop an HIV vaccine have thus primarily focused on recombinant protein vaccines, replication-defective recombinant viral vectors, and DNA vaccines.

The induction of neutralising antibodies is associated with vaccine-induced protection against many viral infections.66 Several lines of evidence suggest that virusspecific CD8-positive T cells, including those specific for HIV,55,67–69 are important in the control of viral replication. The detection of HIV-specific CD8-positive T cells in exposed, uninfected individuals, including infants,70–72 has been cited as additional rationale for the development of vaccines to elicit these; however, these responses have not been consistently detected in cohorts exposed to HIV.54,73 Antigen-specific CD8-positive T cells are most efficiently induced after intracellular antigen expression, which leads to antigen processing and presentation through the MHC class I pathway. Replication-defective recombinant viruses have thus emerged as highly versatile vaccine delivery systems. However, limited replication of some of these vectors—eg, canarypox—or the development of antivector immunity—eg, adenovirus—have limited the immunogenicity of these vectors when administered alone. Immunisation strategies that combine priming with a DNA or recombinant viral vaccine followed by boosting with a heterologous vaccine seem to be particularly effective in generating responses to CD4positive or CD8-positive T cells. Of the replicationdefective recombinant viral vaccines, only canarypox

CD8-positive cells and better understanding of the associations between CD8-cell responses and viral replication or evolution in infants would be helpful in the development of a neonatal vaccine.

Use of neonatal macaque model

Recombinant protein vaccines Monomeric recombinant envelope (gp120) vaccines, designed primarily to elicit HIV-specific antibodies, were among the first HIV vaccines to enter adult and paediatric clinical trials. In PACTG 230, infants were randomised to receive one of two recombinant HIV protein vaccines (Chiron rgp120 with MF59 adjuvant [Chiron Corporation, Emeryville, CA, USA] or VaxGen rgp120 with alum [VaxGen, Brisbane, CA, USA]) at 4, 8, 12, and 20 weeks of age. Both vaccines were tolerated well. Antibody responses 514

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vaccines have been assessed in human infants, but modified vaccinia Ankara (MVA), adenovirus, and adenoassociated virus vaccines are under consideration.

Canarypox vaccines Avian poxvirus-vectored vaccines have also been assessed in infants. PACTG 326 was a phase I trial74 of two different canarypox vaccines (ALVAC vCP205 and vCP1452; sanofi pasteur, Marcy-l’Etoile, France) administered to infants of HIV-infected women at birth and at ages 4, 8, and 12 weeks. The vaccine regimens were well tolerated. vCP205 administered alone did not result in measurable serum antibody concentrations; HIV-specific lymphoproliferative responses (stimulation index ≥3) were detected at two or more timepoints in 44–56% of vaccine recipients, but were not detected in any placebo recipients. HIV-specific CD8-positive T-cell responses were detected by cytolytic assays after in-vitro stimulation of peripheralblood mononuclear cells on at least one occasion in 44–62% of vaccinees. However, detectable responses were of low magnitude (median HIV-specific lysis at an effector-totarget ratio of 25-to-1 was 13–23%) and were repeatedly detected in only two (11%) of the 18 vaccine recipients. In the second phase of this study, vCP1452 was administered with a recombinant gp120 boost (AIDSVAX B/B, VaxGen), with the goal of increasing HIV-specific antibody responses to vaccination. HIV gp120-specific antibodies were detected at 24 weeks after the last immunisation in all infants who received the boosted regimen.75 HIV env-specific lymphoproliferative responses (defined as stimulation index >5 detected at two or more timepoints) were detected in 75% of the group who received ALVAC plus protein boost, but were not detected in any of the infants who received ALVAC alone or placebo.76 HIV-specific CD8-positive T-cell responses were detected only rarely, even after invitro stimulation. Altogether, the modest immunogenicity of the canarypox vaccines in these studies limited enthusiasm for additional studies in infants. In 2005, however, ALVAC-based vaccines were reported58 to confer partial protection against repeated oral challenge of infant macaques with virulent SIVmac251, and infected monkeys had lower viral loads than uninfected monkeys. A limited phase I trial (HPTN 027) of another canarypox vaccine (ALVAC vCP1521) administered with a recombinant gp120 protein boost is planned in Uganda.

Modified vaccinia Ankara MVA, a live, attenuated form of vaccinia, is used as a human vaccine vector because of its restricted replication in human cells, immunogenicity, lack of virulence in animal models (including neonatal or T-cell-depleted animals), and proven safety record in more than 120 000 adults and children as a smallpox vaccine.77 Recombinant MVA vaccines confer protection against measles in infant macaque models.78 DNA-MVA-based vaccine prime-boost regimens are highly immunogenic and can result in a greater than 80% reduction in liver www.thelancet.com Vol 368 August 5, 2006

parasite burden in people challenged with heterologous Plasmodium falciparum.79 However, MVA-fowlpox-based vaccine prime-boost combinations seem to be even more immunogenic and have protected malaria-naive adults against challenge with the liver stage of P falciparum.80,81 Administration of an MVA vaccine expressing a highly conserved mycobacterial antigen (antigen 85A) after previous (0·5–38 years; median 18 years) vaccination with BCG resulted in potent boosting of antimycobacterial T-cell responses.82 An MVA-SIV vaccine has provided limited protection against repeated oral challenge with SIVmac251 in neonatal macaques.58 However, DNA-MVA prime-boost regimens did not result in higher frequencies of memory antigen-specific CD8-positive T cells than DNA vaccination alone in macaques.83 MVA-SIV constructs, administered alone or in combination with DNA-HIV vaccines, protected against challenge with an SIV-HIV chimera (SHIV89.6P84,85). Although the results of a phase I trial on one DNA-MVA HIV vaccine regimen in HIVuninfected individuals indicated poor immunogenicity, boosted CD8-positive T-cell responses were detected in HIV-infected individuals on therapy.86–88 These data suggest that the immunogenicity and efficacy of MVA varies with the vaccine product, the vaccines used in combination with MVA, and the sequence of vaccines given. Several MVA-HIV products are in phase I trials in HIVuninfected adults, including products that express HIV gene products of clade B (Therion Biologics, Cambridge, MA, USA; trial sponsored by the HIV Vaccine Trials Network), or clade C (Therion, Aaron Diamond AIDS Research Center, New York, NY, USA; trials sponsored by International AIDS Vaccine Initiative). A phase I trial of multivalent MVA-HIV vaccines administered with fowlpox-HIV boosts (Therion) in people aged 18–24 years, infected with HIV, and on suppressive antiretroviral therapy, has began to enrol patients through the Pediatric AIDS Clinical Trials Group. Preliminary safety data from these adult studies should be available by the end of 2006 and should facilitate planning for paediatric trials.

Adenovirus vaccines Another group of replication-defective recombinant viral vaccines being studied are the adenovirus-based vaccines. These have elicited potent cellular immune responses when administered alone; a single dose of an adenovirusbased vaccine expressing Ebola virus glycoprotein was protective against viral challenge in a cynomolgus macaque model.89 However, rapid development of antivector immunity has been documented after even a single dose in preclinical primate studies and has resulted in their use in prime-boost regimens. DNA-adenovirus prime-boost regimens seem to be especially promising and are being assessed in adults.90,91 Adenovirus type 5 pre-existing antibodies could compromise the use of this vector in developing countries, where natural infection with adenovirus type 5 is highly prevalent. Since passively 515

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transferred maternal antibodies could potentially interfere with the usefulness of adenovirus type 5 as a neonatal vaccine, seroprevalence studies are being done in pregnant women and young infants. To overcome interference with pre-existing antibodies to the vector, the potential use of other adenovirus strains (adenovirus 35, 11, and chimpanzee) that less commonly cause natural infection is also under investigation. An association between acute primary adenovirus infection, lymphoid hyperplasia, and intestinal intussusception has been reported in children.92

Recombinant adeno-associated virus Recombinant adeno-associated virus (rAAV) has undergone extensive assessment for use in gene-transfer protocols and is under investigation as a vaccine vector. The administration of rAAV-SIV vaccines to macaques resulted in strong humoral and cell-mediated SIV-specific immune responses and suppression of viral replication.93 An rAAV-2 serotype HIV subtype C vaccine is in phase I adult clinical trials; serum antibodies to the AAV-2 serotype were detected in 30% of European, Brazilian, or Japanese children younger than age 10 years and in more than 60% of adolescents or adults, but antibodies to other serotypes were less commonly detected.94 Additional seroprevalence surveys of AAV serotype 1 and 2 are underway in several developing countries. The effects of passively acquired antibodies on the immunogenicity of rAAV vaccines are unknown, but would be a potentially important consideration for their use in neonatal vaccine trials.

DNA vaccines In 1990, Felgner and colleagues95 showed that naked plasmid DNA injected intramuscularly could result in gene expression with subsequent antigen processing and presentation through HLA class I and class II pathways to produce protective CD4-positive and CD8-positive immune responses to the encoded protein. Since then, DNA vaccines have been developed against various pathogens, including HIV.96 Although DNA vaccines are immunogenic in mice and monkeys (including neonates), current vaccines are poorly immunogenic when administered alone to people. Strategies to improve the immunogenicity of DNA vaccines have included optimising gene expression through potent promoters, increasing plasmid doses, Panel: Key questions pertinent to the development of neonatal HIV/AIDS vaccines ● ● ● ● ●

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What are the mechanisms of transmission of HIV via breastmilk? To what extent does cell-free versus cell-associated virus contribute to transmission? What are the characteristics of transmitted viruses? What are the correlates of immune protection? Can infants generate effective immune responses and how can we design immunogens to elicit them? How does HIV sequence variability affect efficacy of HIV-specific immune responses? How can we elicit responses that persist and protect over a lifetime?

formulating the vaccines to limit plasmid degradation and to increase transfection efficiency, co-administration of immunomodulators, and manipulations to enhance cellsurface expression or secretion of encoded proteins. Heterologous prime-boost strategies have shown particular promise in augmenting the immunogenicity of DNA vaccines. DNA-vector prime-boost regimens have been very effective in generating antibody responses in macaques97 and both humoral and cell-mediated responses in people.98 As outlined above, DNA-MVA prime-boost regimens have been assessed in macaques and in people as potential malaria and HIV vaccine strategies. DNAadenovirus prime-boosting strategies seem to be especially immunogenic in macaques.91 Partial control of viral replication after challenge with SIVmac239 was noted in macaques immunised with another DNA-adenovirus prime-boosting regimen;99 control of viral replication in this study was associated with SIV-specific CD8-cell responses and the eventual loss of control with CD8-cell escape. Clinical trials of DNA-adenovirus prime-boost regimens are underway in adults.

Fundamental challenges Basic science questions The Global HIV/AIDS Vaccine Enterprise2 has outlined several scientific priorities. These include: improved understanding of the virological and immunological properties of infecting viral strains; definition of immune correlates of protection against infection or natural disease; and improved understanding of how best to design vaccines to elicit strong neutralising antibody and responses mediated by CD4-positive or CD8-positive T cells. Each of these scientific priorities and those listed in the panel are highly relevant to the development of neonatal HIV vaccines. These include continued definition of key features of the pathogenesis of transmission of HIV via breastmilk (timing, mechanisms) and elucidation of whether infants can generate effective immune responses. Virological and immunological characterisation of infecting strains will be important, using samples from infants in areas with early and mature HIV epidemics. Study of cohorts in different geographical regions would also allow the assessment of potential pathogenic differences between viral clades or recombinants and of host genetic factors—eg, HLA alleles; toll-like receptor or chemokine receptor polymorphisms; polymorphisms in cellular factors important for HIV replication—bearing in mind that antiretroviral treatment is now the universal standard of care for children infected with HIV, as well as for adults. MTCT provides a unique situation in which donor–recipient pairs and the timing of transmission can be easily ascertained. The genetic characterisation of viruses from mother–infant pairs could be particularly informative in the understanding of: whether selective viral variants are transmitted; the diversity of the early quasispecies; how vaccine sequences compare with circulating strains; and the extent to which transmitted www.thelancet.com Vol 368 August 5, 2006

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variants change within individuals or populations over time. Characterisation of changes in viral sequences in individuals and populations, coupled with immuneresponse variables, could provide important data about the sensitivity of transmitted viruses to antibody neutralisation or selective pressures of CD8 cells. The assessment of genetic sequences over time for mutations associated with CD8-positive T-cell escape and correlation with plasma viral load would also provide important evidence about the in-vivo activity of HIV-specific CD8-cell responses in infants. Adaptive immune responses (particularly neutralising antibodies) are probably the main correlates of immune protection for most viral infections and available vaccines.66 Innate responses might also contribute to early resistance or control of infection. Fortunately, new assays and techniques have been developed over the past decade that allow precise measurement and characterisation of innate and HIV-specific adaptive immune responses. Many of these assays can accommodate the small volumes of blood available from infants and will be helpful in characterising HIV-specific immune responses after natural infection or vaccination.50,54

Vaccine trial design Over the next few years, we expect that phase I/II trials of two or three prime-boost strategies will have been completed in adults, paving the way for their use in paediatric studies. The results of phase I trials will be important in establishing the safety and immunogenicity of these products in breastfed infants of HIV-infected women. Since a substantial proportion of transmission of HIV via breastmilk occurs within the first 6 months, phase I/II trials should be designed with the aim of generating protection within 2–3 months of birth. The administration of preventive HIV vaccines, at the same time that routine childhood immunisations are given in infancy, would enhance the feasibility of vaccine delivery; once phase I/II studies have proven the vaccines to be safe and effective in infants, studies to exclude adverse interactions between HIV and other childhood vaccines will be necessary. Efficacy trials could proceed once favourable safety and immunogenicity data from these phase I/II trials are available. The wide-scale implementation of highly active antiretroviral therapy is expected not only to provide virological and clinical benefits to individuals on therapy, but also to reduce their risk of HIV transmission. Plasma HIV RNA concentrations of less than 1700 copies per mL or 1000 copies per mL have been associated with reduced risk of sexual transmission and MTCT, respectively.100 The introduction of perinatal antiretroviral regimens into breastfeeding populations has reduced overall MTCT rates from 25–40% to 8–20%.101 Several postnatal antiretroviral regimens are being assessed for efficacy in preventing breastmilk HIV transmission, but even a reduction in overall transmission rates to 5% would not preclude a www.thelancet.com Vol 368 August 5, 2006

vaccine efficacy trial in breastfed infants. On the assumption that a vaccine would not be associated with an increased risk of acquisition of infection, the estimated sample size for a comparative study between prophylactic antiretroviral and vaccine versus antiretroviral alone in breastfed infants would require about 3500 HIV-exposed infants to document a reduction in MTCT through breastfeeding assessed at 6 months from 5% in infants receiving antiretroviral therapy alone to 3% in the antiretroviral plus vaccine group. However, to address the possibility that a vaccine regimen might result in increased transmission, a sample size of 4200 infants would be necessary. Even the larger sample size is feasible through multicentre collaborative trials.

Ethical and regulatory issues The increasing availability of agents to prevent or treat important paediatric clinical syndromes, as well as the increasing appreciation that growth and maturation could affect the pharmacokinetics, response to, or toxic effects of drugs and biological agents, has led to the prevailing view that paediatric clinical trials are necessary to ensure equal access to novel agents and to continue to advance clinical care of children. In the USA, the Institute of Medicine recently convened a panel of experts to review the ethical conduct of clinical research in children.102 The US Congress, Food and Drug Administration (FDA), and the National Institutes of Health (NIH) have collaborated lately to increase paediatric research, and recent federal statutes require manufacturers of drugs and biological agents to undertake paediatric studies to ensure that new products are appropriately labelled for children. Efforts are underway worldwide to increase paediatric research efforts and to define guidelines for the inclusion of children in research. Children are vulnerable, both biologically and socially. As such, any research study in children must incorporate safeguards.103 US federal regulations and international guidelines generally require that risks to participants be reduced to a minimum and be reasonable in relation to the expected benefits. Robust systems to protect research participants provide a good foundation for protecting participants in paediatric research. However, additional resources should be committed to the development of ethical and legal standards that specifically protect children. The development of these standards and paediatric research protocols depends on the participation of a diverse group of individuals with expertise in the scientific, psychosocial, and ethical aspects of paediatric clinical care and research. For any new drug or biological agent, preliminary safety studies in adults are generally required before the initiation of paediatric studies. Since some infants might already be infected before vaccination—ie, those with in-utero infection—early adult studies of vector-based vaccines should include their assessment in immunocompromised animals and HIV-infected (as well as uninfected) adults.

For the US Congress, FDA, and NIH research and statutes see http://www.fda.gov/cder/ pediatric/#prea

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For WHO guidelines on care, treatment, and support for women living with HIV/AIDS and their children in resourceconstrained settings see http://www.who.int

The precise requirements for preclinical (in particular, reproductive toxicology studies) and adult testing can vary according to the product, but paediatric trials should not be delayed until an agent has been fully assessed in adults. Antiretroviral agents have successfully entered clinical trials soon after preliminary adult studies opened, and paediatric vaccine trials should ideally follow this model. An essential point in the undertaking of paediatric HIV vaccine trials is that all other preventive and therapeutic measures that apply to the study population should be offered to trial participants. For example, standard-ofcare antiretroviral treatment should be offered to pregnant women who are HIV positive for their own health and to prevent MTCT. Maternal access to appropriate antiretrovirals during gestation should be continued postpartum; standard-of-care drugs should also be provided to infants diagnosed with HIV who participate in vaccine trials. This approach might increase the number of required participants, but vaccine studies will remain eminently feasible. Informed consent is the cornerstone of good research practice. A successful neonatal vaccine regimen to prevent transmission of HIV via breastmilk will probably depend on the initiation of immunisations at birth and an accelerated vaccine schedule over the first 2–3 months of life. Informed consent to begin treatment could be difficult to obtain during labour or delivery. Completion of the informed consent process before delivery has greatly facilitated the enrolment of newborn children into previous trials of antiretroviral treatment and HIV vaccines. Liability concerns are often raised as a possible obstacle to HIV vaccine trials in children. In the USA, for licensed and recommended childhood vaccines, the Vaccine Injury Compensation Act provides liability protection for vaccine manufacturers and compensation for children who might have been harmed by vaccination. There is no comparable system for investigational vaccines, and most large pharmaceutical companies self-insure, an approach that could prove difficult for small biotech companies and non-governmental organisations involved in the search for an HIV vaccine. Governmental indemnification insurance might provide a means to remove this potential barrier.

Laboratory support The Global Vaccine Enterprise Scientific Strategic Plan emphasises the need to establish a global system of laboratories—vaccine immune monitoring centres—to collaborate in characterising HIV-specific immune responses in the context of natural infection or vaccine trials. Priority activities for these laboratories include: the development of a wide range of standardised assays to allow comparison across and between natural history, preclinical, and clinical studies; the development and sharing of necessary reagents; and continuous quality assurance. A set of so-called core or reference laboratories will be charged with assay development, standardisation, 518

training, and quality assurance along with the undertaking of specialised assays (particularly those requiring specialised equipment). Satellite laboratories at clinical study sites will process blood specimens, do basic virological or immunological assessments, and ship specimens for more specialised assessments to core laboratories. The development of a parallel but integrated consortium of laboratories focused on studying the pathogenesis of breastmilk HIV transmission and the characterisation of infant immune responses to natural infection or vaccines would greatly facilitate the development of neonatal vaccines. Paediatric scientists should be integrated into the laboratory decision-making process of the Global Vaccine Enterprise. Wherever possible, protocols, laboratory space, personnel, reagents, and quality-assurance mechanisms should be shared. However, paediatric investigators might need to develop reagents uniquely suited for studies of neonates—eg, panels of infecting viral strains. Furthermore, early diagnosis of HIV infection in infants is crucial within natural history and vaccine studies, but few assays are available that can reliably diagnose young infants in limited-resource settings. The development and standardisation of diagnostic assays for use in field settings is important. Finally, paediatric investigators will probably need to adapt or prioritise assays owing to the limited blood volumes available from infants and confirm the usefulness of these assays. For example, ELISPOT assays have been used in many paediatric studies to detect antigen-specific T cells because of the very small blood volumes needed for the assays. Available data66 suggest that ELISPOT assays that measure interferon γ secretion give a general indication of the immunogenicity of a vaccine, but that the results do not necessarily indicate protective immunity. The development and validation of assays that allow more thorough assessment of responder-cell phenotype—eg, expressing CD4 or CD8—and function than presently possible are needed. Although multiparameter flowcytometry-based assays seem promising, the large blood volumes and specialised equipment needed could hinder their application to large-scale paediatric studies. Emerging technologies—eg, chip-based MHC-peptide arrays—could be especially useful for such highthroughput paediatric studies.

Conclusions The development of a safe and effective paediatric preventive HIV vaccine would be an extremely important advance and would have a major effect on control of the HIV/AIDS pandemic. Unique features of MTCT and paediatric HIV disease could be especially helpful in elucidating correlates of immune protection and could facilitate rapid assessment of vaccine efficacy. Vaccine responses primed during infancy could provide the basis for lifetime immunity to HIV. We therefore propose that www.thelancet.com Vol 368 August 5, 2006

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international vaccine initiatives, including the Global Vaccine Enterprise, should include the development of preventive paediatric HIV vaccines as a priority. Paediatric scientists should be integrated into the leadership and activities of the Global Vaccine Enterprise. Adult and paediatric investigators should collaborate closely on preclinical and clinical vaccine development, particularly to ensure the timely order of studies in adults and children. The development of a consortium of laboratories focused on studying the pathogenesis of transmission of HIV via breastmilk and the characterisation of infants’ immune responses to natural infection or vaccines would greatly facilitate the development of neonatal vaccines. This consortium of paediatric laboratories should be integrated into the network of vaccine immune monitoring centres; sharing of laboratory space, personnel, equipment, protocols, reagents, and qualityassurance programmes could provide economy of scale.

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Conflict of interest statement We declare that we have no conflict of interest.

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Acknowledgments We thank Patricia Fast, Executive Director, Medical Affairs, International AIDS Vaccine Initiative, for very helpful discussions. K Luzuriaga was supported by funding from the US National Institutes of Health (grants NIH/NIAID R01 AI032391 and NIH/NICHD K24 HD01489, which was not involved in study design, data interpretation, writing of the Review, or in the decision to submit this paper for publication. The Ghent Working Group on HIV in Women and Children (KL, ML-N, FD, JLS are members) also provided funding. References 1 Klausner RD, Fauci AS, Corey L, et al. Medicine: the need for a global HIV vaccine enterprise. Science 2003; 300: 2036–39. 2 The Global HIV/AIDS Vaccine Enterprise. The Global HIV/AIDS Vaccine Enterprise scientific strategic plan. PLoS Med 2005; 2: 0111–21. 3 UNAIDS. 2004 report on the global AIDS epidemic. http://www. unaids.org/bangkok2004/GAR2004_html/GAR2004_00_en.htm (accessed Jan 31, 2006). 4 Mofenson LM. Advances in the prevention of vertical transmission of human immunodeficiency virus. Semin Pediatr Infect Dis 2003; 14: 295–308. 5 Connor EM, Sperling RS, Gelber R, for the ACTG 076 Study Group. Reduction of maternal-infant transmission of human immunodeficiency virus type 1 with zidovudine treatment. N Engl J Med 1994; 331: 1173–80. 6 Cooper ER, Charurat M, Mofenson L, et al. Combination antiretroviral strategies for the treatment of pregnant HIV-1-infected women and prevention of perinatal HIV-1 transmission. J Acquir Immune Defic Syndr 2002; 29: 484–94. 7 European Collaborative Study. Mother-to-child transmission of HIV infection in the era of highly active antiretroviral therapy. Clin Infect Dis 2005; 40: 458–65. 8 Guay LA, Musoke P, Fleming T, et al. Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: HIVNET 012 randomised trial. Lancet 1999; 354: 795–802. 9 Jackson J, Becker-Pergola G, Guay L, et al. Identification of the K103N resistance mutation in Ugandan women receiving nevirapine to prevent HIV-1 vertical transmission. AIDS 2000; 14: F111–15. 10 Eshleman S, Mraca M, Guay L, et al. Selection and fading of resistance mutations in women and infants receiving nevirapine to prevent HIV1 vertical transmission (HIVNET 012). AIDS 2001; 15: 1951–57. 11 Cunningham CK, Chaix ML, Rekacewicz C, et al. Development of resistance mutations in women receiving standard antiretroviral therapy who received intrapartum nevirapine to prevent perinatal human immunodeficiency virus type 1 transmission: a substudy of Pediatric AIDS Clinical Trials Group protocol 316. J Infect Dis 2002; 186: 181–88.

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