The expansion of vaccine technologies over the last two decades has provided new tools and knowledge that can be applied to RSV vaccine development. Here we consider subunit, live attenuated viruses, and gene-based vector approaches. Whole inactivated virus, no matter how it is adjuvanted, is going to face major regulatory hurdles in RSV-seronegative infants and will not be discussed as an option.
To achieve vaccine-induced protective immunity, effector mechanisms should be present early after exposure to mitigate the viral evasion of innate immune mechanisms. This would be best accomplished by the presence of neutralizing antibody. In addition, if effector T cells were present more rapidly after infection, viral clearance would potentially be accomplished earlier. This would result in less pathology, because fewer host cells would be lost and the total viral antigen load would remain lower and be less likely to induce excessive T-cell responses that could interfere with gas exchange and damage tissue. Presumably, this would result in less illness during the infection, less repair, and fewer long-term consequences for airway architecture and function.
There are several potential goals for RSV vaccine development, and the characteristics of the vaccine and study designs will vary depending on target population. The ideal and ultimate goal for a RSV vaccine would be to protect neonates from infection during the first season and eliminate the large burden of hospitalization that occurs in the 2-3-month-old age group. The only immunization strategy that has been evaluated in 1-2 month-old infants utilized live attenuated virus. Virus attenuation was achieved by cold-adaptation and chemical mutagenesis and ultimately by transferring selected mutations into infectious molecular clones. Additional modifications were empirically derived by deleting selected nonessential genes (NS1, NS2, M2-2, or SH) or adding additional attenuating mutations not necessarily associated with temperature sensitivity (87
). These live viruses were evaluated in immunodeficient mice and chimpanzees to achieve a rank order of attenuation, then tested in adults, seropositive children (>2 years of age), and seronegative children, before evaluation in infants (<6 months of age). Two vaccines have been evaluated in seronegative infants. The first (cpts248/404) caused a high frequency of nasal congestion and was considered to be insufficiently attenuated (88
). Nasal congestion was counted if it interfered with eating, sleeping, or resulted in obligate mouth-breathing. The other virus (rA2cpts248/404/1030/ΔSH) was evaluated in 32 infants at one of two doses (104.3
pfu or 105.3
pfu), and 12 placebo recipients (89
). The low and high dose inoculations resulted in 63% and 94% infection rates with 2.4 log10
pfu/ml and 3.5 log10
pfu/ml peak titers in nasal secretions, respectively. This was about 2 log10
lower than cpts248/404 and reduced the frequency of nasal congestion to 19% and 44% for the 2 dose levels tested. The most striking result of this study was that the frequency of virus shedding after a second vaccine inoculation 4-8 weeks later was only 29% and 44% for the low and high doses with much lower peak virus titers. While there was evidence of infrequent F- and G-specific IgA antibody responses in immunized infants, they were not sufficient to correlate with protection. Live-attenuated virus vaccine approaches are discussed in more detail below.
Protecting infants from 6 months to 2 years of age from their first or second RSV infection could also have a major impact on morbidity. There is a high attack rate in this age group and RSV remains a common cause of hospitalization and doctor visits up to the age of 5. Some of the advantages of focusing on this age group include: (i) waning of maternal antibody to RSV or delivery vehicles; (ii) they are past the stage of idiosyncratic apnea events; (iii) maturation of immune system including capacity for somatic mutation and development of high affinity antibodies has occurred; (iv) children in this age range are a common source of RSV transmission to younger infants and parents; and (v) lower frequency of breastfeeding makes nasal delivery more feasible.
Immunization of persons older than 2 years of age implies that vaccination will be done in the setting of pre-existing immunity, since nearly everyone has been infected by that age. Children and adolescents who have been infected multiple times with RSV with diminishing clinical symptoms are less likely to benefit from RSV immunization than infants. However, there are two major subgroups of adults that should be considered as target populations for RSV immunization. First are pregnant women or young women expecting to become pregnant. Immunization of pregnant women, particularly those with deliveries anticipated in the last quarter of the calendar year, could potentially boost antibody levels and improve the effectiveness of passively transferred immunity in the newborn. Immunization of pregnant women is controversial and is a complex topic that cannot be adequately covered here. Vaccinating women of child-bearing age prior to pregnancy is a potential option but would require vaccines that could induce a durable boost of pre-existing antibody levels. One of the major biological hurdles for immunizing young women involves the property of RSV to repeatedly infect healthy adults. Vaccination would have to be significantly better than natural infection in terms of magnitude and durability of immune responses to have a measurable difference on levels of maternally derived RSV-specific antibody.
Adults >65 years of age represent another target population that should be considered in RSV vaccine development. Immunization of elderly persons faces the problems imposed by pre-existing immunity described above and has the added challenge of the aging immune system. While RSV does influence morbidity and mortality in the institutionalized elderly, the pathogenesis is different than the bronchiolitis of primary infection and invariably involves underlying cardiopulmonary dysfunction. In that sense, RSV may be more of a triggering event than the direct cause of the pathology leading to illness in this age group. This would potentially make the threshold for protective immunity more difficult to achieve. There are several laboratory challenges for conducting RSV vaccine studies in adults. One is that everyone has pre-existing immunity, so all immunogenicity measurements will have to be compared to background levels. This significantly complicates the identification of immune correlates. Secondly, the diagnosis of RSV in adults is more difficult than in children experiencing primary infection. Rapid antigen detection and culture are less likely to be positive in RSV-infected adults, so the infection endpoint requires nucleic acid detection by PCR (90
Antigen selection for a RSV vaccine, as for any viral vaccine, requires consideration of the protective antigenic sites, antigenic diversity, genetic variability, and the immunological effectors targeted for induction, toxicity, or immunomodulatory properties, and feasibility of production or expression. For live-attenuated virus vaccines, in which most if not all antigens are included, it is still important to know how attenuation has affected expression of the desired antigenic determinants and avoided expression of antigens that may cause toxicity or adversely impact vaccine-induced immune responses. If RSV antigens are produced from gene-based vectors, the fact that RSV has an exclusively cytoplasmic replication cycle is important to consider. The native genes have never had to adapt to the nuclear environment. If the delivery vehicle initiates transcription in the nucleus like DNA plasmids or recombinant adenovirus vectors, the RSV gene will have to be extensively codon-modified or transcripts will be rapidly degraded. If the vector has a cytoplasmic replication program like alphavirus or poxvirus vectors, codon modification is less critical.
RSV F is a trimer of 70 kDa heterodimers. It is a type I integral membrane glycoprotein and mediates pH-independent membrane fusion from without during viral entry and cell-to-cell spread. RSV F is similar to HIV-1 gp160, ebola GP, and other paramyxovirus fusion protein that require furin cleavage to expose a hydrophobic peptide and assume the fusion-active conformation (91
). The RSV F glycoprotein is surface-expressed and has at least three identified major antigenic sites associated with neutralizing antibodies (92
). The known neutralizing epitopes in RSV F are all located in the region between the 2 heptad repeats (HR) in the major cleavage fragment F1. One of these sites is the epitope for palivizumab, a licensed monoclonal antibody (mAb) that significantly reduces RSV-associated hospitalizations when passively administered to infants at risk for severe disease (93
). Therefore, F is a known protective antigen and a target for neutralizing antibody and is considered the most important antigen for including in a RSV vaccine. F is highly conserved between strains, even between strains from the A and B subtypes. F-specific neutralizing antibodies to antigenic sites A (~aa255-275) and C (~aa422-438) are cross-protective. RSV F is unique among fusion proteins, because it has two furin cleavage sites that liberate a 27 amino acid peptide containing 2 of the 5 N-linked glycosylation sites. The biological functions of this peptide have not been fully defined, although a homologous peptide from bRSV (termed virokinin) can interact with tachykinin receptors and cause smooth muscle contraction (94
). Like G, RSV F also has heparin-binding domains (95
). In addition, F has been shown to interact with the TLR4/CD14 complex and can induce IL-6 production in monocytes (96
). The potential biological effects of the 27 aa peptide, heparin-binding, TLR interaction, and other biochemical aspects of RSV F need to be considered in vaccine design and evaluation.
The RSV G glycoprotein is a 90 kDa type II integral membrane protein that has 4 N-linked glycosylation sites and is also heavily O-glycosylated. RSV G has been associated with viral attachment and is sometimes referred to as the attachment glycoprotein. It is also the target of known neutralizing antibodies, and G is the most variable of RSV proteins, suggesting that it is under immune pressure (91
). Variability in the G glycoprotein is the major distinction between the major RSV subtypes. For these reasons, G is a candidate antigen to be considered in vaccine design. Interestingly, G-deleted viruses can still infect cells in vitro
, indicating that G is not required for attachment and entry (97
), although the absence of G does diminish infectivity in vivo
. This suggests a potential role for G in immune evasion or immunomodulation, which should be considered in vaccine design. RSV G has a chemical composition consisting of ~30% serines and threonines and ~10% pralines, which is unusual for viral glycoproteins. The closest counterpart is the ebola GP, which has a mucin-like domain that appears to cap the receptor binding domain prior to pH dependent cleavage by cathepsin L (98
). RSV G has an alternative initiation codon at amino acid 48 in the transmembrane domain, which results in significant shedding of soluble G from infected cells (99
). The soluble form of G is thought to function as a decoy for neutralizing antibody (100
). The mucin-like domains are located on both ends of the protein and around a nonglycosylated central domain that has 4 conserved cysteines organized as a cysteine noose followed by a heparin-binding motif (101
). A ‘CXXXC’ sequence can interact with the fracktalkine receptor (CX3CR1) (103
), and the propensity for G binding to glycosaminoglycans and C-type lectins provide multiple potential mechanisms for G to affect immune functions. Decisions to include G as a vaccine antigen merit additional investigation into the potential biological effects RSV may have on the immune response.
Internal (non-surface exposed) antigens are highly conserved between RSV strains and are known to be a source of many T-cell epitopes (104
T-cell responses are thought to be important for RSV clearance because of the severe and progressive disease that occurs in patients with selected immunodeficiency syndromes and supportive data from animal models. CD8+
T cells also have favorable immunomodulatory properties (67
). While certain CD4+
T-cell responses have been associated with vaccine-enhanced disease, they have value for amplifying antibody responses and other T-cell effector mechanisms. Therefore, in gene-based vectors including chimeric viruses, if a broader set of T-cell responses is desired, proteins such as N, M, or M2 could be considered as additional vaccine antigens, in combination with F and G. The SH (small hydrophobic) protein of RSV has surface exposure and is thought to possibly function as pentameric ion channel (106
). Prior efforts to immunize with this protein have not successfully induced neutralizing antibody and because of its limited size does not contain many potential T-cell epitopes. Therefore, it has not been considered to be a high priority vaccine antigen. The NS1 and NS2 (nonstructural) proteins have significant immunomodulatory activity and, even though they have been viewed as potential therapeutic targets, have not been strongly considered as vaccine antigens. The large polymerase protein (L) and phosphoprotein (P) have not been studied carefully for their potential value as vaccine antigens.
Vaccine delivery platforms
Vaccine antigens have traditionally been delivered by infection with attenuated live virus or injection of whole inactivated virus. Fifteen of the 17 licensed antiviral vaccines fall into one of those categories, and the other two are virus-like particles (VLPs). The successful development of VLPs for hepatitis B (HepB) and human papillomavirus (HPV) belies the slow pace in achieving new licensed vaccines based on the significant advances in molecular biology over the last 30 years. It is likely that vaccines for RSV, and other viral pathogens with difficult biological properties like HIV and herpes simplex virus, will require the application of new technology platforms, in addition to a greater depth of understanding disease pathogenesis and mechanisms of immunity (107
Live-attenuated viruses and vectors
Although whole inactivated RSV would be difficult to advance again into clinical evaluation because of safety concerns, vaccination with live attenuated RSV given parenterally or mucosally can be done safely. Early trials of live RSV given intramuscularly were unsuccessful because of poor immunogenicity but were not associated with signs of aberrant immune responses or enhanced illness. Therefore, much effort has gone into the development of live attenuated candidate RSV vaccines. The leading candidate viruses are based on attenuating mutations that cause temperature sensitivity discovered during in vitro
cold-adaptation. The development of a system to construct infectious molecular clones of RSV (108
) has allowed the introduction of these and other selected mutations into precisely engineered constructs and the production of highly characterized attenuated vaccine strains. Some of these viruses have been evaluated in seronegative infants (1-2 months of age) and have been shown to partially protect against a second dose of the vaccine strain as noted above. This approach has the advantage of utilizing most of the antigenic content of RSV, and the proteins should be expressed in their native conformations. Since it is delivered nasally, it should induce local immunity in the respiratory tract, which may be important for protection against RSV. A live attenuated molecular clone (MEDI-559) is now in Phase II clinical evaluation in children (5-24 months of age) and infants (1-3 months of age). The live attenuated virus approach has the following challenges, particularly if it is targeted for neonates prior to their first infection. (i) The virus needs to be sufficiently attenuated to avoid significant disease from the primary infection caused by vaccination and retain sufficient replication capacity to generate a protective antigen exposure. Nasal congestion is problematic in infants during breast- or bottle-feeding. (ii) The therapeutic window (between the thresholds for efficacy and symptoms) needs to be achieved in a large majority of infants, erring on the side of safety. This is challenging because of the wide variations in developmental status, levels of transferred maternal antibody, MHC alleles, and innate susceptibility to infection among infants under 2 months of age. It will also be difficult for an attenuated virus to achieve better immunity than infection with wildtype virus, which does not provide solid protection from reinfection. (iii) Idiosyncratic reactions, particularly apnea, in this age group may be associated with a vaccine delivered in the airway whether perceived or real. (iv) RSV is difficult to grow to high titer, has a relatively high ratio of defective to replication-competent particles, is relatively fragile to freeze-thaw, and vulnerable under most storage conditions. These features will complicate the manufacturing, lot-to-lot consistency, and distribution of a live-attenuated RSV vaccine. Delivery of live RSV intramuscularly is another approach to attenuation and avoids many of the challenges listed above. However, the issues noted under point (iv) would still apply, and the earlier failure of live virus delivered parenterally may have been in part due to the low dose levels achieved. The ultimate attenuation would be a replication-defective RSV that can only express viral antigens from the initial transduced cell.
Chimeric viruses and replication-competent gene-based vectors
An approach that retains the advantage of replication in the respiratory tract but avoids most of the noted challenges is the use of chimeric viruses. Selected relevant genes from RSV have been expressed in related paramyxoviruses like bovine parainfluenza (PIV), Newcastle disease virus (NDV), or Sendai virus (SeV) that can be delivered via the respiratory tract. These viruses are attenuated because their native tropism is not human. Therefore, replication is limited in vivo
, but in vitro
replication properties are more favorable than RSV for manufacturing. Also, the frequency of pre-existing immunity against these viruses is very low, so the impact of maternally derived antibody will be negligible. Another version of this approach is to express RSV antigens from replication-competent vectors like adenovirus serotype 4 (Ad4), vesicular stomatitis virus (VSV), or BCG, for either mucosal or parenteral delivery. Each of these vectors has unique properties that may provide advantages or challenges depending on the target age group, vaccine antigen, and goals of immunization, but space limitations do not allow a full discussion here. Bovine PIV3 expressing the RSV F and the hPIV F (MEDI-534) is the only candidate product in this category that has advanced to clinical trials. Data have only been reported for seropositive children in whom it appeared to be safe. However, there was no viral shedding and no significant immunogenicity. This construct replicated to relatively high titers in RSV-naive AGMs (~5.5 and ~7 log10
pfu/ml in nasal secretions and BAL, respectively) and protected them from challenge with wildtype RSV, even though serum neutralizing antibody titers remained relatively low (~1:16) (74
Replication-defective gene-based vectors
There are a number of vector systems derived from nucleic acids, viruses, or bacteria that can deliver a gene encoding the vaccine antigen of choice. These systems all have nuances involving promoters, tropism, delivery options, durability of expression, gene insert size, immunostimulatory properties, vector-specific immunity, and methods of manufacturing that are important to consider. Vector systems of note in this category including adeno-associated virus (AAV), vesicular stomatitis virus, herpesviruses, poxviruses, naked DNA, and encapsidated DNA will not be discussed because of space limitations. Alphavirus vectors will be discussed briefly, and recombinant adenovirus vectors (rAd) will be discussed in more depth. Replication-defective vectors need to have robust manufacturing capacity, because achieving sufficient antigen expression for immunization depends on the vaccination dose. In most cases, delivery is parenteral, because it is difficult to achieve enough antigen production from a single round of expression when delivered mucosally. A major advantage of this approach is that gene expression should mimic natural infection resulting in authentic protein structures to elicit relevant antibody responses and proteasomal processing and MHC class I antigen presentation for CD8+ T-cell induction. Other advantages of this approach include the improved safety profile because of lack of replication capacity, avoidance of immune evasion strategies by selective gene expression, targeted antigen delivery based on selected vector tropism, and the ability to select vectors with low seroprevalence to avoid pre-existing anti-vector immunity.
Alphavirus vectors based on Venezuelan equine encephalitis virus (VEE), Semliki forest virus (SFV), or Sindbis virus have all been successfully used to express vaccine antigens (109
). They are particularly attractive as vector systems because of the self-amplifying RNA replicon that has the potential to significantly increase antigen expression levels. They merit special consideration for RSV vaccine development, because they have an entirely cytoplasmic transcription program, which means codon-modification requirements will be less restrictive. In addition, murine experiments showed that parenteral immunization with recombinant VEE could uniquely elicit mucosal IgA responses to the vaccine antigen (110
), which would be relevant to the airway-restricted RSV. VEE vectors expressing RSV F have been evaluated in mice and cotton rats and demonstrated immunogenicity, protection, and immune response patterns that do not include Th2-like responses (111
). Therefore, this platform technology is a candidate for clinical evaluation. The major limitations of this vector platform have been manufacturing capacity and translation of immunogenicity profiles from mice to primates.
rAd, particularly serotype 5, expressing HIV envelope glycoproteins have been extensively studied in clinical trials and have been shown to be well tolerated and immunogenic (112
). The rAd5 vector was initially designed for gene therapy, but because of induction of vector-specific immune responses and rapid clearance, its value as a vaccine exceeded its usefulness for gene therapy. One of the most attractive features of the rAd vector platform is the robust manufacturing capacity with yields ranging above 1013
particle units from a single 10 liter bioreactor production run. There are 51 recognized serotypes of human adenoviruses divided into 6 species (A-F). Ad5 is a species C virus and uses the coxsackievirus adenovirus receptor (CAR) through binding to Fiber and an integrin co-receptor that binds an ‘RGD’ sequence on the Penton base (115
). In humans, CAR is widely expressed in gap junctions between epithelial cells and myocytes and is also expressed on human erythrocytes (116
). The biology underlying the reason rAd5 is more immunogenic for both induction of antibody and CD8+
T-cell responses than other rAd vectors has not been explained, but is the subject of intensive investigation because of 3 specific concerns associated with rAd5. The first is a historical association with a highly publicized adverse outcome in a gene therapy study (117
). This was a case in which an extremely high dose of rAd5 was injected intravenously directly into the liver of a person with abnormal liver function. The extreme antigen load led to an acute inflammatory response, thought to be related to an antigen-induced ‘cytokine storm’, and the death of the patient. While this case was tragic and a valid reason for caution, it is not directly relevant to the use of modest doses of a vaccine vector given intramuscularly, and subsequent clinical trials of candidate rAd5 vaccines have been conducted without serious adverse events related to vaccine-induced inflammation. A second concern arose following the STEP trial (118
) in which a rAd5 vector expressing the Gag, Pol, and Nef genes from HIV-1 was evaluated in individuals at high-risk of HIV-1 infection. In this study, among MSMs (men who have sex with men), vaccinees had a higher rate of HIV-1 infection than placebo recipients. The higher infection rate was associated with men who were uncircumcised and tended to be greater in those with pre-existing immunity to Ad5. While the basis for vaccine-induced increased infection has not been fully explained, this outcome has created a stigma for rAd5 that has been difficult to overcome, although it is not relevant to the purpose of protecting infants from RSV. The third major limitation for rAd5 is the relatively high Ad5 seroprevalence in the general population which is ~50% in North America and can be >90% in some developing countries (119
). Pre-existing immunity to rAd vectors has been shown to diminish the magnitude and frequency of T-cell responses to the vaccine antigen and to a lesser extent blunts the vector-induced antibody responses (112
). Therefore, the rAd5 vector platform for adult populations may have diminished efficacy because of pre-existing Ad5 immunity. The effect of pre-existing immunity can be mitigated by priming immunizations, so rAd5 could still be considered as a booster in a heterologous vector combination regimen. In the case of RSV, there may be a window of opportunity to use rAd5 vectors. Children between 6 months (after maternally derived antibody has waned) and 2 years of age are Ad5-seronegative (120
). Therefore, targeting that age group with rAd5 vectors expressing RSV antigens would be most advantageous.
Alternative rAd vectors have been constructed using species B (serotypes 14 and 35) and D (serotypes 26 and 28) human adenoviruses (122
), chimeric adenoviruses in which fiber genes or immunogenic domains from hexon are swapped with rare serotype viruses (123
), and from adenoviruses derived from nonhuman primates (124
). These vectors have been developed primarily because of their relatively low seroprevalence and engineered to be as immunogenic as possible. For immunization against RSV in the presence of maternal antibody (under the age of 4 months) these rare serotype rAd vectors may have advantages over rAd5. Since adenovirus genes are expressed in the nucleus, the sequence of RSV genes inserted into any rAd vector will need to be carefully codon-optimized to ensure optimal expression.
Immunization with a single purified protein is one of the simplest possible vaccine platforms. However, except in the case of HepB surface antigen or HPV L1 that both assemble into VLPs, subunit vaccines have not been successfully licensed for any viral vaccine product. Purified RSV F glycoprotein (PFP-2) formulated in alum has been evaluated in clinical trials in adults. It was found to be well tolerated and modestly immunogenic. A single injection of 50 μg in alum in pregnant women at 30-34 weeks of gestation only induced a >4-fold rise in serum neutralizing activity in ~10%, but antibody to F in babies and in breast milk was significantly higher among the vaccinees (125
). A single dose of this same vaccine in healthy elderly subjects (>60 years of age) elicited >4-fold rise in serum neutralizing activity against either RSV A or B subtype in 61% (126
) and in frail elderly in 47% that was relatively stable for about 6 months. Another product consisting of a mixture of F, G, and M was also tested in subjects >65 years of age. In this study, single doses of 25, 50, and 100 μg were given with alum or a 100 μg dose was given without alum. Surprisingly, the non-adjuvanted product induced the highest antibody responses, and 58% developed a >4-fold increase in serum neutralizing activity (127
). It is intriguing to consider the possibility that this antigen assumed some VLP structures, since it has been shown that matrix (M) protein from other paramyxoviruses is sufficient to form VLPs (128
). Chimeric VLPs based on the Newcastle disease virus M and containing RSV G have been shown to be immunogenic in mice (129
). Another subunit product based on the central conserved region the G glycoprotein (aa130-230) was constructed as a fusion protein with an albumin-binding domain from streptococcal protein G, produced in prokaryotic cells and formulated with an alum-based adjuvant (Adjuphos). Despite promising results in murine studies, studies in adults showed relatively low capacity for inducing neutralizing activity (130
), and challenge studies in rhesus macaques showed no reduction of viral load, vaccine antigen-specific induction of IL-13, and detectable eosinophilia in lung (131
Because of the legacy of vaccine-enhanced disease associated with FI-RSV, there are significant regulatory hurdles for advancing subunit proteins into seronegative infants. Given as a protein, even with modern adjuvants, subunit protein vaccines will elicit primarily CD4+ T-cell and antibody responses. Relative to live attenuated virus or gene-based vectors, there will not be a significant CD8+ T-cell response. Not having the positive influences of viral clearance and modulation of CD4+ T cells away from Th2 CD4+ T-cell responses provided by CD8+ T-cell responses creates additional theoretical concerns about the potential for vaccine-enhanced disease when immunizing RSV-naive infants. These concerns would have to be weighed against the quality, magnitude, and durability of the neutralizing antibody induced by the candidate subunit product. It is possible that tertiary and quaternary structural features of the proteins or the immunostimulatory properties of a VLP are necessary to achieve the specificity, breadth, and magnitude needed for protective antiviral responses. The RSV F glycoprotein is thought to exist as a trimer on the surface of the virus like other class I fusion proteins. Preservation of the antigen sites that are targeted by neutralizing antibodies, the oligomeric state, and other structural aspects of F or G in addition to adjuvant selection should be thoughtfully considered if they are produced as purified proteins for candidate vaccines.
Recently, the structures of the epitope (aa254-277) recognized by palivizumab and motavizumab (132
) and another epitope (427-437) recognized by the antibody 101F (McLellan et al
., manuscript submitted) have been solved. These data suggest that even more limited subunit vaccines could be envisioned, expressing just the structures of key neutralizing epitopes. Building distinct epitope structures onto other protein scaffolds has been developed conceptually (133
) and piloted in HIV vaccine design with the gp41 2F5 epitope (Ofek et al
., manuscript submitted). This structure was able to elicit structure-specific antibody, but the antibody was unable to neutralize HIV-1 isolates. One potential derivative of producing a scaffolded epitope is that a vaccine antigen could be constructed that induced antigen-specific antibody against the structure without inducing antigen-specific T-cell responses since the linear sequence required for a T-cell epitope is not required for recreating the structure. Interestingly, palivizumab, motavizumab, and 101F antibodies bind trimeric F protein with much higher affinity than they bind peptide, even though they are described as linear epitopes. This and historical data from other peptide immunization studies suggest that the determinants for antibody recognition and binding of an epitope are more complex and include a greater surface area than just that between the linear peptide and the combining region of the antibody. Therefore, the concept of building a vaccine response one epitope at a time will require new breakthroughs in our understanding of what defines an epitope and the structural basis of antibody-epitope affinity.