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SUPPLEMENT ARTICLE Increasing Complexity of Vaccine Development Stanley A. Plotkin University of Pennsylvania, Doylestown Vaccines already developed have been enormously successful. However, the development of future vaccines requires solution of a number of immunologic problems, including pathogen variability, short effector memory, evoking functional responses, and identification of antigens that generate protective responses. In addition, different populations may respond differently to the same vaccine because of genetic, age, or environmental factors. Keywords. immune memory; correlates of protection; influenza; pertussis; HIV; rotavirus; dengue; cytomegalovirus; respiratory syncytial virus. The success of vaccines is counted as one of the crowning achievements of medical science, with enormous reduction in mortality and morbidity rates [1]. Quoting the first edition of the Vaccines textbook, “The impact of vaccination on the health of the world’s peoples is hard to exaggerate. With the exception of safe water, no other modality has had such a major effect on mortality reduction and population growth” [2] (Table 1). One of the great successes of vaccination has been the eradication of smallpox. This was possible because smallpox was almost always clinically apparent and thus contacts of a case patient could be vaccinated at exposure. Another success has been the great reduction in the incidence of polio, made possible through the use of 2 vaccines: the inactivated and oral forms. As is well known, Hilary Koprowski developed the first oral polio vaccine, although the strains developed by Albert Sabin ultimately were used worldwide. The 2 vaccines are complementary, in that the inactivated vaccine always induces serum antibodies that protect against paralysis, and the oral vaccine renders the vaccinee less likely to excrete poliovirus. Nevertheless, despite the remarkable successes achieved with vaccines, for example against measles, rubella, invasive pneumococcal Presented in part: Hilary Koprowski Memorial Symposium, The Wistar Institute, Philadelphia, 27 June 2014. Correspondence: Stanley A. Plotkin, MD, Vaxconsult, University of Pennsylvania, 4650 Wismer Rd, Doylestown, PA 18902 ([email protected]). The Journal of Infectious Diseases® 2015;212(S1):S12–16 © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected]. DOI: 10.1093/infdis/jiu568 S12 • JID 2015:212 (Suppl 1) • Plotkin disease, meningitis, and cervical cancer, there are also many current failures to develop badly needed vaccines. This article considers some examples of such failures and describes the scientific issues responsible for them. Table 1 lists some of the key problems. The first is pathogen variability and escape from vaccine-induced immune responses. This problem limits the efficacy of influenza vaccines. Although both inactivated and live vaccines are available, the former is less effective in infants [3] and the latter less effective in the elderly [4], but the major problem is that both types are less effective because the circulating viral hemagglutinin often changes from that used to make the vaccines. Consequently, both vaccines are far from completely effective. A 2012 meta-analysis [5] showed that influenza vaccine is less than optimal even in older children and young adults (Table 2). Its efficacy could be improved in several ways. The first way, namely, the addition of a second type B lineage to make quadrivalent vaccines, has already been applied. Increasing the dose of hemagglutinin improves antibody responses and therefore efficacy, as does the incorporation of adjuvants into the vaccine. Adding neuraminidase, which is better conserved than hemagglutinin, might increase efficacy. However, the optimal improvement would be to add conserved epitopes to the vaccine. The epitopes might come from the viral nucleoprotein (T-cell epitopes), the M2e protein, or, most interestingly, the stalk of the hemagglutinin. The recent identification of a conserved epitope far from the variable head of the hemagglutinin might allow production of a more effective vaccine that would not have to be changed each year [6]. Table 1. Key Problems in Vaccinology Table 3. Transvaccinology Better diagnosis Pathogen variability and escape Short effector memory Antigenic changes in current strains Increased toxin production of current strain Obtaining the right functional response Antibodies not bactericidal; efficacy against infection lower than against disease Lower efficacy for acellular than for whole-cell pertussis vaccine (waning antibodies, lower cellular immunity, or fewer virulence antigens) Postvaccination immunity wanes faster after acellular pertussis vaccine Population specific challenges Pathogen-specific problems Uncertain correlates of protection Unknown antigen(s) required for protection A second key problem, a rather common one for vaccines, is short effector memory. This problem is well illustrated by the recent resurgence of pertussis [7]. Pertussis vaccines made by inactivating whole bacteria were effective in controlling disease but were also reactogenic and were therefore replaced by acellular vaccines containing 1–5 pertussis proteins. Although the acellular vaccines solved the reactogenicity issue, pertussis became resurgent after those vaccines went into standard use in developed countries. Various hypotheses have been advanced to explain the resurgence (Table 3). Although strain change may play a role, the principal problem is the rapid waning of antibodies, particularly to the pertussis toxin. Current efforts are directed towards prolongation of immune responses through the use of adjuvants or more virulence proteins from Bordetella pertussis [8]. However, an extended duration of effectiveness would be desirable for many vaccines, particularly those consisting of subunit proteins. It is the common dogma that antibodies protect by neutralizing entry by viruses into cells and by killing pathogenic bacteria. A means to induce broadly neutralizing antibodies to human immunodeficiency virus (HIV) before exposure has bee the subject of intensive research, but with limited success [9]. Four large phase III trials of AIDS vaccines have been done, but only one has shown some success [10], the one conducted in Thailand using a canarypox vector carrying the gene for HIV envelope as prime, followed by the viral envelope protein as boost (Table 4). Protection against acquisition was about 30%, although it was higher early in the trial and then faded, and it was also higher in low-risk than in high-risk subjects [11]. The great surprise was that the correlate of protection that emerged was unrelated to neutralizing antibodies. The major correlate of protection was antibodies against the V1–V2 loops of the virus functioning as antibody-dependent cellular cytotoxic antibodies [12], particularly of the immunoglobulin G3 class [13]. This finding has also been seen with some other viruses, against which nonneutralizing antibodies also may have protective functions [14]. The canarypox used for priming in the Thai trial may also be key to the moderate success of vaccination, because it induces a proinflammatory cytokine milieu. Vaccine efficacy is also influenced by population-specific factors. For example, rotavirus vaccines consisting of attenuated viruses given orally have been highly effective against infantile gastroenteritis in developed countries, but in poor tropical countries their effectiveness is greatly reduced [15] (Table 5). What is the explanation for this difference? Possibilities include high levels of maternal transplacental antibodies, neutralization by antibodies in breast milk, interference by simultaneous oral polio vaccine administration, malnutrition, differences in the intestinal microbiome, and interference by other intestinal pathogens. The reasons are incompletely understood and are probably multiple, but some evidence indicates roles for maternal antibodies and changes in the intestine due to infections [16]. A vaccine against dengue is a high priority in view of the spread of mosquito vectors. Neutralizing antibody against the Table 4. Table 2. Adultsa History of AIDS Vaccines Trials Efficacy of Influenza Vaccines in Children and Young Vaccine Type Dates Influenza Strain Efficacy, % H1N1 74–82 H3N2 B 40–58 51–58 H1N1 86–90 H3N2 B 75–90 44–70 Inactivated Live a Reasons for Pertussis Resurgence Data from a meta-analysis [5]. Antigen 1998–2003 Envelope gp120 2005–2008 Adenovirus 5 vector carrying gag 2004–2010 2010–2012 Concept Result Antibody protection Cellular immunity No efficacy Canarypox vector carrying envelope, envelope boost Prime boost 31% efficacy DNA for envelope, adenovirus 5 vector carrying envelope boost Prime boost Negative efficacy Complexity of Vaccine Development • Negative efficacy JID 2015:212 (Suppl 1) • S13 Table 5. Rotavirus Vaccine Against Severe Disease in Tropical Countries Vaccine RV1 RV5 Country Efficacy, % Brazil 77 Malawi South Africa 49 77 Nicaragua 77 Kenya Ghana 83 65 Vietnam 73 Bangladesh 46 envelope protein has been thought to be protective, but a recent efficacy trial has cast doubt on that idea [17]. The vaccine in the trial was a quadrivalent live vaccine in which the replicating virus was the yellow fever 17D attenuated vaccine strain into which the RNA expressing envelope and premembrane proteins for dengue types 1, 2, 3, or 4 were inserted [18]. The anticipation was that each virus would replicate and induce neutralizing antibodies against all 4 serotypes, and indeed early studies showed that to be the case [19]. Nevertheless, when the vaccine was tested for efficacy in Thailand, although high efficacy was shown against types 3 and 4, efficacy against type 2 was not seen, and efficacy against type 1 was only moderate. Several explanations seemed possible, listed in Table 6. Interestingly, recent data showed that the structure of dengue virus differs according to the temperature at which virus replication takes place. In cell culture or in the human at 37°C the structure of the virus is expanded, whereas at lower temperatures in the mosquito the particle is more compact [20]. This could mean that epitopes exposed on the vaccine virus are not exposed on the mosquito challenge virus, which is therefore able to enter cells without being neutralized. However, another explanation for which there is now evidence is that only viral replication induces homotypic antibodies (N. Jackson, personal communication). In the quadrivalent vaccine the type 4 chimera replicates well and induces homotypic antibodies to itself but only heterotypic antibodies to dengue type 2. The type 2 virus does not replicate Table 6. Possible Explanations for Low Efficacy of Chimeric Dengue Vaccine Higher challenge dose of type 2 or strain variation, so more antibodies needed Rapid infection of monocytes by dengue type 2; antibody thus not effective T-cell response also needed for protection Type 2 antibodies heterotypic, not homotypic Chimera presents of envelope protein in different conformation Structural differences between virus injected by mosquitoes and envelope in chimeric virus S14 • JID 2015:212 (Suppl 1) • Plotkin and therefore does not induce homotypic antibodies. The difference between homotypic and heterotypic antibodies may involve differences in epitopic specificity crucial to protection. Because human cytomegalovirus (CMV) is the predominant infectious cause of congenital abnormalities since the elimination of rubella as well as the most common infectious complication of organ and hematogenous stem cell transplantation, a vaccine against CMV is badly needed [21–23]. The first CMV vaccine to be extensively tested in humans was a live attenuated virus developed in my laboratory [24]. This strain, called Towne, was immunogenic after subcutaneous injection but did not establish latency in vaccinated individuals. Thus, it was safe but unfortunately in human trials it failed to prevent acquisition of wild CMV by women exposed to infected children [25]. However, when administered to renal transplant recipients who lacked prior immunity to CMV and who received a kidney from a seropositive donor, the vaccine greatly reduced severe disease [26]. This was the first proof of concept that a vaccine could influence CMV infection. During that work it was found that an important antigen for the generation of neutralizing antibodies is a surface glycoprotein called gB [27]. Accordingly, a vaccine was developed consisting of gB together with MF-59, an oil in water adjuvant [28]. This product was immunogenic after 3 doses, but antibodies fell precipitously thereafter. Importantly, during the first 18 months of a controlled trial vaccinees had about a 60% reduction of CMV acquisition [29]. Even more striking, the same vaccine reduced viremia in kidney or liver transplant recipients by >80% [30]. These results were gratifying, but a new discovery changed our thinking on CMV vaccines: the identification of a pentameric complex of proteins on the surface of the virus, which was shown to induce the majority of neutralizing antibodies in human immune globulin. The complex had previously been unnoticed because neutralization tests were done in fibroblast cells, but CMV can also enter epithelial and endothelial cells by a different mechanism, mediated by the pentameric complex [31]. So it seemed that vaccine developers had been working with only one of the important CMV proteins [32]. This complex, composed of the glycoproteins gH and gL together with proteins UL128, 130, and 131, is the subject of intense efforts to incorporate it into next-generation CMV vaccines [33]. Many candidate CMV vaccines are in development (Table 7). Respiratory syncytial virus has long been recognized as the most important respiratory pathogen in infants and second in importance only to influenza in the elderly. Attempts in the 1960s to develop vaccines against respiratory syncytial virus have been frustrated first by a disaster with an inactivated whole virus vaccine that exacerbated disease in vaccinees, second by difficulties in sufficiently attenuating the virus for use in infants <6 months of age, and third by relatively poor immunogenicity of the fusion protein that induces neutralizing antibodies. Recently, structural biology has come to the rescue of Table 7. CMV Vaccines in Development Table 8. Unsolved Problems in Vaccinology Vaccine Type Problem Live Short immune memory More stimulus of Tfh cells and stronger induction of innate immunity by Toll-like receptor agonists Multiplicity of virulence antigens in complex pathogens Conserved epitopes Vaccines containing multiple epitopes Structural biology to identify exposed structures Finding correlates of protection in protected vaccines Immaturity and postmaturity of immune system Systems biology to identify unregulated genes Addition of cytokines or neutralize cytokines needed for immune responses Use of nanoparticles to be taken up by M cells Attenuated strain (Towne) Recombinants with wild virus (Towne-Toledo) Alphavirus replicon to generate viruslike particles or RNA Pox virus, adenovirus, lymphocytic choriomeningitis virus vectors carrying CMV antigens Replication – defective virus Nonlive Recombinant gB glycoprotein with adjuvant DNA plasmids Peptides Viruslike particles Abbreviation: CMV, cytomegalovirus. this field by demonstrating a prefusion form of the F protein on the virus that is highly immunogenic [34]. In addition, adenovirus vectors carrying the F protein are showing promise [35]. Another example of the need to find the right antigens is the search for a vaccine against group B meningococci. Excellent vaccines have been available for years against the other major serogroups but not against group B because of homology between the capsular polysaccharides and neural glycoproteins. However, the development of genomics, or so-called reverse vaccinology, permitted the identification of 4 proteins that induce bactericidal antibodies against most group B strains [36, 37]. The combination of those proteins with outer membrane vesicle from the organism has been licensed in Europe, and effectiveness data are awaited [38]. Although the bacille Calmette-Guérin strain of Mycobacterium bovis has been an effective live vaccine against tuberculosis in childhood, its effectiveness against adult tuberculosis is questionable. Therefore, a new vaccine is sought. Although candidate vaccines have been developed based on bacille Calmette-Guérin with added genes from Mycobacterium tuberculosis, viral vectors carrying those genes, and even attenuated M. tuberculosis strains, efficacy has been unconvincing [39]. The basic problem is the absence of a known mechanistic correlate of protection; in other words, the protective immune response is unknown, although it has long been thought to be a T-cell function [40]. Despite induction of T-cell responses, a candidate vaccine recently failed to protect in a clinical trial [41]. For a malaria vaccine, the problem is the complexity of the life cycle and the multiplicity of antigens that would be needed to give complete protection. Nevertheless, it is clear that the sporozoite contains antigens that do give protection, notably the circumsporozoite antigen, and that protection is mediated by both B-cell and T-cell responses [42, 43]. Injection of Mucosal immunization with nonreplicating antigen Possible Solution Adjuvants capable of selectively expanding cell types: dendritic, B, Th1, Th2, Th17, CD4+, CD8+, or Treg cells Use of single or combined Tolllike receptor ligands directed to specific cell types Difficulty in generating T-cell immunity without replicating vaccines Addition of adjuvants that stimulate T cells Abbreviations: Tfh, follicular T-helper; Th, T-helper; Treg, regulatory T. whole inactivated sporozoites has shown high efficacy [44]. The task now is to add other sporozoite antigens to improve on the moderate protection thus far achieved [44, 45]. Stepping back and considering vaccinology as a branch of immunology, one is forced to realize that there are many unsolved problems, including immune memory, multiplicity of protective antigens for some pathogens, multiple HLA types among vaccinees, the need for conserved epitopes, identification of correlates of protection, immaturity of the germinal centers in the newborn, immunosenescence of T cells in the elderly, and insufficient stimulation of mucosal immunity. Table 8 suggest some pathways to solve these problems during the coming years. In conclusion, vaccinology has made great strides in the past but is now attempting to deal with infections that are complex and host factors that mitigate efficacy. Fortunately, the advances in molecular biology, structural biology and systems biology offer concrete opportunities to solve these difficult problems, but new vaccines will be the fruit of difficult labor. Hilary Koprowski would have been happy to face these intellectual issues, and it is a pity that he is no longer with us. Note Potential conflict of interest. The author is a consultant to many vaccine manufacturers, including those attempting to develop the vaccines discussed. The author has submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Complexity of Vaccine Development • JID 2015:212 (Suppl 1) • S15 References 1. van Panhuis WG, Grefenstette J, Jung SY, et al. Contagious diseases in the United States from 1888 to the present. N Engl J Med 2013; 369:2152–8. 2. Plotkin ST, Plotkin SA. A short history of vaccination. In: Plotkin SA, Orenstein WA, Offit PA, eds. Vaccines. 6th ed. Philadelphia, PA: Elsevier/Saunders, 2013:1–13. 3. Lee BY, Shah M. Prevention of influenza in healthy children. Expert Rev Anti Infect Ther 2012; 10:1139–52. 4. 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