A call to cellular and humoral arms

[Human Vaccines 4:2, 148-157; March/April 2008]; ©2008 Landes Bioscience

Commentary

A call to cellular and humoral arms Enlisting cognate T cell help to develop broad-spectrum vaccines against influenza A Julie A. McMurry,1,* Bert E. Johansson1 and Anne S. De Groot1,2 1EpiVax,

Inc.; Vaccines Division; Providence, Rhode Island USA; 2Brown University; Providence, Rhode Island USA

UT E

.

Abbreviations: MHC, major histocompatibility complex; HA, hemagglutinin; NA, neuraminidase; CIV, conventional inactivated virus vaccine; LAV, live attenuated vaccine; CTL, cytotoxic T lymphocyte; Th cell, T helper cell

OT D

IST

Hemagglutinin (HA) and neuraminidase (NA) are the major surface glycoproteins of the influenza virus.6 Humoral immunity against these two proteins is the primary means of resistance to and recovery from influenza virus infection.6,7 Antibody to HA can neutralize viral infectivity; antigenic variation in this molecule is responsible for frequent outbreaks of influenza and for the poor control of infection by immunization.7,8 Antibodies to NA are infection-permissive across a broad range of NA-antibody levels, but they result in the reduction of pulmonary virus titers below a pathogenic threshold.7-9 Secretory IgA and IgM provide protection against the establishment of initial infection, however, IgG neutralizes newly replicating virus once infection has been established.10,11 In addition, IgG prevents viral pathology in the (murine) lung. For both primary and secondary immune responses,10,11 T helper cells play a critical role in isotype switching (the generation of IgG) and in affinity maturation (the generation of high-affinity antibodies).12 T-helper (Th) cells are also required for CTL-mediated clearance of the virus in an ongoing infection. Immune responses to the more highly conserved internal proteins, M1, M2 and NP, have been studied with focus on the cross-reactive cytotoxic T cell (CTL) and T-helper responses.13-15 M1 and NP also elicit antibody responses;16,17 however, these antibodies have been demonstrated not to play a significant role in protection.18,19 Production of influenza antigens in baculoviral, yeast or other recombinant systems provide an opportunity to modify the protein to increase immunogenicity, to direct the immune response toward a set of antigens and/or to modify vaccine formulations through the use of adjuvants. Modification may be needed for recombinant proteins to increase immunogenicity.20,21 Antigenic variation. Two distinct types of antigenic variation occur in influenza viruses: antigenic drift and antigenic shift; both types pose significant challenges to the immune system and to the design of influenza vaccines. Antigenic drift consists of relatively minor alterations at the genetic level that generally result in gradual antigenic changes. Antigenic shift involves major changes in HA and/or NA. Antigenic drift. Antigenic drift in both HA and NA occurs as a result of point mutations that result in amino acid changes.

SC

IEN

CE

.D

ON

Influenza A is an important cause of morbidity and mortality worldwide. In the United States alone influenza kills 30,000 to 50,000 people in a non-epidemic year and significantly more in an acute epidemic.1 An emerging pandemic influenza virus, such as H5N1, could have a devastating economic and social impact. The Surgeon General estimates that at least 43 million Americans, especially those younger than 1 and older than 60, are at risk of death from influenza. Antigenically distinct influenza virus strains emerge regularly, mandating changes in influenza vaccine antigenic composition. Consequently, the immunity engendered by the conventional influenza vaccines is relevant only for a short time. However, by incorporating conserved influenza T cell epitopes, it may be possible to develop more immunogenic, broader-spectrum vaccines that may be efficacious over a longer period. This review summarizes the critical components of effective influenza vaccines, a rational vaccine design approach, and the pertinent influenza immunology.

RIB

Keywords: Influenza, T cell, B cell, cognate, help, epitope

BIO

Influenza Antigens

©

20

08

LA

ND

ES

Influenza remains a pervasive public health problem in spite of the wide availability of two currently licensed vaccines against influenza, both of which are dominated by the immune response to a single highly variable antigen, hemagglutinin (HA).2-4 To be effective, an influenza vaccine must be designed and manufactured prior to the beginning of an influenza season or pandemic, and must induce immune responses that recognize the circulating strain.5 Through global surveillance, the human influenza strains that pose the greatest risk for infectious spread can be chosen and influenza vaccines can be formulated accordingly. However, such vaccines are susceptible to failure since significant antigenic variation can occur in the time elapsing from the selection of the vaccine candidate strain and wild-type virus exposure.5 *Correspondence to: Julie A. McMurry; EpiVax, Inc.; 146 Clifford St. Providence, Rhode Island 02903 USA; Tel.: 401.272.2123; Fax:.272.7562; Email: JAM@ EpiVax.com Submitted: 01/31/07; Revised: 10/03/07; Accepted: 10/14/07 Previously published online as a Human Vaccines E-publication: www.landesbioscience.com/journals/vaccines/article/5169

148

Human Vaccines

2008; Vol. 4 Issue 2

Enlisting T-help for influenza vaccines

OT D

IST

RIB

UT E

.

a virus-like-particle influenza vaccine recombinantly produced in baculovirus (Novavax, Gaithersberg, MD); these vaccines also focus on the generation of antibody responses. Conventional inactivated virus vaccines. Conventional inactivated virus vaccine (CIV) is derived from formalin—inactivated, high-yield reassortant viruses whose internal genes are from a A/PR/8/34 high-yield donor parent;18 its HA and NA are derived from recently prevalent influenza A and B viruses predicted to cause widespread infection. CIV is standardized according to the HA antigenic content as defined by single radial immunodiffusion (SRID) testing. The protective efficacy of CIV against influenza virus infection has been 70–90% in studies of vaccinated military personnel, but protection of persons in high-risk categories (e.g., the elderly) has been variable (0–80%).33 A 2006 Cochrane review of 64 studies published between 1974 and 2004 found that in homes for the elderly (with good influenza vaccine match and high viral circulation) the effectiveness of vaccines against influenza—like illness was only 23% (95% confidence interval 6% to 36%) and non-significant against influenza (RR 1.04: 95% confidence interval 0.43 to 2.51).34 How effective are current influenza vaccines in induction of protection against challenge with a heterovariant virus? Conventional vaccines are effective only if the HA of the predicted vaccine strain is closely matched in antigenic structure to the HA of the circulating strain. During the 2003–2004 influenza season, 12.7% of clinical influenza isolates were antigenically similar to the vaccine strain A/ Panama/2007/99 (H3N2), and 87.3% were similar to the drift variant A/Fujian/411/2002 (H3N2); a high incidence of influenza due to the A/Fujian strain in people vaccinated with A/Panama was reported.35 The serologic response to influenza virus vaccine also varies with the age of the recipient and previous exposure to influenza virus.36 In primed individuals (those who have had previous exposure to influenza), vaccination with CIV induces “protective” levels of antibody against HA, which prevent infection in 89% of recipients shortly after vaccination. In contrast, in unprimed recipients, only 65% developed protective levels of serum anti-HA antibodies.37 In general, levels of anti-HA antibody are low after a single dose of vaccine but increase significantly in response to a second dose. The higher efficacy of the vaccine in primed individuals is likely due to the induction of greater B-cell memory—due in part to cross-reactive helper T-cell populations induced during prior infection and vaccination.38-40 T-cell response may be a critically important factor in the response to new HA antigens associated with antigenic shift; we posit that vaccines against newly emerging (eg. pandemic) influenza viruses should be designed with this in mind. Live attenuated vaccine. Like CIV, the LAV vaccine is derived from a master donor virus (MDV) strain containing temperature sensitive (ts), cold-adapted (ca) and attenuation (att) mutations in several genes coding for internal proteins41 and contains the HA and NA from a vaccine candidate strain. How effective is LAV in preventing new infections? Despite evidence that vaccines containing whole killed or live attenuated virus induce cross-reactive T cells in humans42 and mice,43 reinfection with homologous or heterotypic virus occurs. Proponents of the LAV claim that the live virus vaccine induces a more broadly cross-reactive immunity than CIV.44 The reported 86% efficacy rate of live virus vaccines is

Current Influenza Vaccines

IEN

CE

.D

ON

Consequently, antigenic variants within a subtype emerge and are gradually selected as the predominant virus while the preceding virus is suppressed by specific antibodies arising in the population. While both HA and NA are highly susceptible to antigenic drift, Kilbourne et al.22 determined that, over a thirty year period, HA evolved more rapidly than did NA. NP and M1 drifted even more slowly than did the surface glycoproteins.23 Although a single amino acid change in HA or NA is sometimes associated with a significant antigenic change,24 it may have little effect on the total antigenic properties of these antigens.25 Nonetheless, mutations occur sequentially during the spread of a virus, such that a change in a single epitope may engender some selective advantage.22 Significant change from an epidemiological viewpoint generally requires changes to accumulate in two or more sites.26 Antigenic shift. Two mechanisms, recombination and adaptation, have been proposed to explain the major HA or NA changes known as antigenic shift. Genetic reassortment can occur between human viral strains or between human virus and animal influenza viruses.27 In 1957 and 1968, new influenza viruses emerged from reassortment with swine and avian viruses, respectively. The descendants of these viruses continue to cause the majority of influenza infections in humans. Taubenberger et al.28 described a second mechanism for antigenic shift, when they demonstrated that the influenza virus associated with the 1918 pandemic did not originate through a reassortment event involving a human influenza virus: all eight genes of the H1N1 virus are more closely related to avian influenza viruses than to influenza from any other species, suggesting that an avian virus must have infected humans and accumulated mutations that allowed it to spread from person to person. Thus, pandemic influenza may originate through direct spread and adaptation of a virus from animals to humans.

©

20

08

LA

ND

ES

BIO

SC

The two currently licensed vaccines against influenza are the conventional inactivated virus vaccine (CIV) and the live-attenuated vaccine (LAV). Since circulating influenza strain HA and NA can vary, a new type of vaccine based on the prevalence of circulating strains (and their HA and NA proteins) is made available as often as every year, if necessary. These reformulated versions of influenza vaccine are not always protective—the CDC estimates the influenza vaccine efficacy to be 70–90%, but only when the HA of the vaccine strain is well matched to the HA of the circulating virus.29,30 The conventional inactivated vaccine (CIV) is delivered intradermally or intramuscularly and the live attenuated, cold-adapted vaccine (LAV) is given intranasally. The commercially available CIV is a trivalent vaccine containing HA from three different circulating strains of influenza. CIV elicits good serum antibody responses but poor mucosal IgA antibody and less robust cell-mediated immunity.31 LAV may possibly elicit a long-lasting, broader immune response, which more closely resembles natural immunity.31 While the LAV vaccine was implemented during the 2003–2004 season in the US,32 the safety profile of the LAV contraindicates its use in those at greatest risk for becoming ill with and transmitting influenza—young children and older adults. Other vaccines that have been studied in humans include virosome—adjuvanted influenza vaccine (Inflexal V; Berna Biotech, Berne, Switzerland) and www.landesbioscience.com

Human Vaccines

149


Table 1

Incorporation of conserved T cell help into vaccines offers three important advantages: immunogenicity, broad cross-reactivity, and safety

(CIV) Conventional Inactivated Virus

(LAV) Live-Attenuated Recombinant M2e Virus HA-only Stand-alone

ICS (Th epitope) Peptide cocktail

ICS protein cocktail or VLP

- (Th prime) - (Th prime) ++++

++++

Immunogenicity Serum Ab +++ ++ ++ Mucosal Ab + +++ + Antigenic Breadth +/- + -

+ (Anti-M2e) - (Anti-HA/NA) + (Anti-M2e) - (Anti-HA/NA) -

+ +

+ ++

- -

+++ +++

++

?

++++

++++

Safety for elderly / immune-compromised

++++ ++++

++++

+++

RIB

Safety

++++ ++++

UT E

T Help within subtype T Help across subtypes

++++

.

Cross-reactivity

+++

Recently, more attention has been directed toward M2 protein as a vaccine antigen. M2 has a 23 amino-acid non-glycoslyated ectodomain referred to as M2e which has limited variation most likely due to the fact that the M2 open reading frame is embedded within the M1 domain.16,57 The antibody response to M2e in humans16 and mice57 was poor after infection. However, several studies have demonstrated in animal models that antibodies toward M2e can be induced by vaccination with a stand-alone M2e vaccine which induced an immunity that did not prevent infection but restricted viral replication, mitigated illness and reduced deaths after an experimental infectious challenge.58 Immunity engendered by M2e is similar to immunization with viral NA. Both are infection-permissive and therefore likely to produce a definitive and cross-protective immunity after subsequent infectious challenge. Please see Table 1 for a summary of advantages and disadvantages of conventional and experimental vaccines.

©

20

08

LA

ND

ES

BIO

SC

IEN

CE

.D

ON

not, however, significantly different from the 60 to 80% effectiveness rate achieved by immunization with inactivated vaccine.44,45 Additionally, LAV is labeled for use only in healthy individuals 2 to 49 years of age; therefore, it cannot be used in patients most at risk for influenza morbidity and mortality. Although, a recent study in children demonstrated clinical superiority of the LAV over CIV46 more studies are needed to confirm the clinical efficacy and possible superiority of LAV over CIV. Experimental vaccines. There are several novel vaccines against influenza currently under development, a thorough discussion of which is beyond the ambit of this review; therefore, our discussion will be limited to two leading candidates: recombinant HA-only (rHO) produced in baculovirus, and M2e. Continuing studies of a purified uncleaved HA vaccine (rHO) produced in baculovirus, have shown that recombinant production in baculovirus obviates the need for egg-grown virus and a high-yield reassortant. However, the rHO vaccine is three-fold less immunogenic than conventional vaccine20,21,47 and is effective only if the HA of the vaccine strain is closely matched antigenically to the expected wild-type strain HA.48 As demonstrated during the 2004–2005 influenza season, shortages of influenza vaccine provoked national concern and a call for longer-lasting influenza vaccines.49,50 In November 2006, the WHO further recommended “immediate and sustained action and funding” for these development efforts.29 Because experimental H5N1 vaccines have demonstrated moderate to poor immunogenicity at the highest doses,20,51 the NIAID has put new emphasis on research into adjuvants and dose sparing techniques. Studies of experimental vaccines produced in response to H5N1, H9N1 and H2N2 outbreaks in Hong Kong unfortunately suggest that a dose >15 ug of HA, boosting, and/or adjuvants may be required to achieve protective titers.52-55 According to influenza experts including Gregory Poland, “a variety of candidates—including vaccines administered with adjuvant, peptide-based vaccines, and vaccines developed with adenovirus vectors—deserve consideration in order to meet the terrible threat of pandemic influenza.”56

OT D

IST

A cocktail of immunogenic consensus sequence (ICS) Th-cell epitopes could be used as a prime to augment the immunogenicity of conventional vaccines and assist with rapid recovery from influenza infection. A vaccine comprised of ICS-optimized antigens HA, NA and internal proteins would possess the cognate T help required to elicit protective high-affinity antibodies specific for HA and NA. Both of these ICS approaches (protein and peptide) would also generate a critical population of T helper cells cross-reactive with emerging virus strains, whether antigenically drifted or shifted.

150

Correlates of Protection Both humoral and cellular immune responses are important in the host defense to influenza infection.19,59 Antibodies have been shown to be critical for virus neutralization and for protection against subsequent homologous infection. T cells play a role in the clearance of the virus in an ongoing infection; memory T cells to conserved epitopes have also been shown to confer protection to heterologous infection.60,61 Because both antibody and CTL responses are Th cell dependent, the activation of Th cells is critically important to the magnitude and kinetics of the host antiviral immune response.62 In the absence of functional (memory) CD4+ T cells, the rate of viral clearance upon secondary infection slows considerably, beyond the degree seen in the primary response.63-65 B cell recognition. Antibody response is the first line of defense against influenza; neutralizing antibodies can effectively inhibit binding to cell-surface receptors for HA. Several neutralizing antibody epitopes have been identified in HA, including HA 92–105, 127–133 and 183–195 of influenza virus H3N2. Antigenic variation within these B cell epitopes in a circulating strain can dramatically limit vaccine

Human Vaccines


OT D

Figure 2. Antigenic sites of hemagglutinin (HA) and neuraminidase (NA). The filled circles on each glycoprotein represent the sites of antibody binding. These sites are highly variable and are localized around the proteins’ active sites, represented by oval discs.

Similar approaches to those described above have been used to a lesser extent to study the antigenic properties of NA.25 The catalytic site, a large pocket of the distal surface formed by the tetramers, is surrounded by antigenic variable regions.74-76 See Figure 2 for an illustration of these regions. Effect of antibody response to influenza. Neutralizing anti-HA antibody presumably acts by preventing attachment of the virus to host cells or by interfering with the fusion event subsequent to endocytosis.77 Antibody to NA exerts its effects at a later stage of multicycle infection and is therefore infection-permissive over a broad range of antibody concentrations. Anti-NA antibody may cause: (1) steric inhibition of enzyme function, thereby delaying viral release from the host cell; (2) cross-linking virions resulting in aggregation of viral particles, thus reducing the number of effective infecting units; (3) cementing of free viral particles to host cell-associated NA antigen; and, (4) inhibition of final detachment of the budding virion.78 Through both vaccination and infection, it is also possible to generate humoral responses to the more highly conserved internal proteins, M1, M2 and NP.18,79 However, antibodies to these proteins do not appreciably impact the course of infection. Despite a poor antibody response against M2 protein following infection in humans16 and mice57 a dedicated M2e vaccine induced an immunity that did not prevent infection but did restrict viral replication, reduced illness and deaths after an experimental infectious challenge.58 For conventional inactivated split vaccines, HA and NA subunits are not purified to the absolute exclusion of other proteins; thus, low levels of M1-, M2- and NP- specific T cells are generated. However, these T cells can provide only bystander help to the vaccine since the HA and NA are phyically dissociated from the other proteins.80,81 T-cell recognition. All T cells recognize pathogen-derived, linear peptide sequences presented in the context of the MHC on the surface of antigen presenting cells (see Fig. 3A). MHC haplotypes are highly polymorphic within and between species. In contrast to

ON

Figure 1. The ten constituent proteins of Influenza A. Hemagglutinin trimers and neuraminidase tetramers are the major surface antigens of influenza encoded by the HA and NA genes respectively. The M protein gene, differentially processed, yields both the M1 matrix protein and the M2 ion channel. Nuclear protein (NP) provides a scaffold upon which the viral RNA genes can be fixed for transcription and translation by the polymerase complex comprised of proteins PB2, PB1 and PA.

IST

RIB

UT E

.


©

20

08

LA

ND

ES

BIO

SC

IEN

CE

.D

efficacy. B cell response to NA is also important; addition of NA to HA vaccine formulations increases immunogenicity, improves protective efficacy against homotypic infection, and accelerates viral clearance in heterotypic infection.30,66 However, the level of immunogenicity of the NA within CIV and LAV vaccines varies considerably.67 Properties and locations of B cell epitopes. While the ability to predict B cell epitopes would clearly be useful, the computational sequence analysis tools required to achieve this are still early in development. Locating the major antigenic sites on a given HA molecule usually involves two complementary techniques: (1) generation of an antigenic map by using a panel of monoclonal anti-HA antibodies to select viral mutants expressing antigenically—changed HA molecules, and (2) analysis of the three dimensional structure of HA molecule and a comparison of the amino acid sequence of the HA from related and mutant strains. Carbohydrate moieties at certain sites could result in additional differences in antigenicity observed among HA strains. Antibodies to influenza, including neutralizing antibodies, react with conformational determinants—some of which are present only in the trimeric form of HA68 (see Fig. 2). Anti-HA antibodies have also been raised to linear determinants: several groups have synthesized short peptides corresponding to sequences in the HA molecule and have tested the ability of these peptides to induce antibodies.69-71 Most antibodies raised to the peptides did not cross-react with antigenic sites on the HA; those that did cross-react did not reduce viral titers or mitigate disease in vivo.70,71 HA1, the membrane-interacting domain of hemagglutinin, is highly conserved at the sequence level, thus antibodies to this region have been shown to cross react between some subtypes.72 Unsurprisingly, antibodies to less-well conserved domains do not cross-react except for strains within a subtype.73

www.landesbioscience.com

Human Vaccines

151

LA

ND

ES

BIO

SC

IEN

CE

.D

ON

OT D

IST

RIB

UT E

.


©

20

08

Figure 3. (A) In order to induce naïve B cells to produce antibodies to a protein antigen and to initiate an adaptive immune response, several events must be coordinated, usually within specialized regions of secondary lymphoid organs (e.g. lymph nodes, spleen, etc.). The first step in this process is the nonspecific internalization of the antigen by professional antigen-presenting cells (APC), such as dendritic cells (lower left panel). The mature APC process the antigen into peptides that are then presented to a naïve T cells in major histocompatibility complex (MHC) class II molecules on the APC surface. The interaction of the T-cell’s receptor (TCR) with this MHCII:peptide complex is identified as T-cell Signal 1. In order to fully activate the T-cell, this must be accompanied by additional signals from what are termed “co-stimulatory molecules”, such as CD80, CD86, etc. provided by the APC (T-cell Signal 2). In the absence of such a second signal, T cells may become anergic. Once fully activated, these CD4 T cells divide and can produce an array of cytokines with manifold activities. An interaction between B-cell IgM and IgD receptors and cognate antigen is required to initiate activation of the naïve B-cell (lower middle panel). This is termed B-cell Signal 1. The B cell internalizes and processes the antigen:Ig complex and presents cognate T cell epitopes in the context of MHCII on its surface. T helper cells, upon encountering the cognate epitope:MHC complex, deliver cytokines and engage CD40L (B-cell Signal 2/3); this drives the B-cell to activate, proliferate and differentiate into memory B cells and antibody secreting plasma cells (lower right panel). Thus, full activation of a B-cell and its corresponding T-cell is dependent on (1) proper presentation of the T-cell epitope derived from the protein antigen by a DC and a B-cell, and (2) recognition of the peptide/MHC complex by the TCR. Cytokine signals from the T-cell initiate a cascade of further immunostimulatory events that cause B cells to expand, switch isotypes and undergo phenotypic changes resulting in the establishment of memory B cells. (B) As illustrated in this figure, a complete lack of T cell epitopes would eliminate cognate T help and lead to B cell apoptosis.

152

Human Vaccines



OT D

IST

RIB

UT E

.

TH cells directed against HA, NA and the internal proteins M1 and NP.106,107,97 In humans and mice, most of the TH clones found to be reactive across subtypes were specific for M1 or NP.98,99 Synthetic peptides corresponding to sub-regions of HA can stimulate TH cells that react to HA within a subtype; some of these TH cells are also able to react to HA across subtypes.100,101 Cognate T help. Cognate T cell help is critical to the quality and kinetics of the anti-HA and anti-NA antibody response, both primary and secondary.2,13,14,102-104 Cognate help is characterized by the requirement that epitopes recognized by Th cells be covalently linked to epitopes recognized by the responding B cells (Fig. 3). Interestingly, both human and murine TH cells specific for internal components M and NP are able to collaborate with B-cells in both HA- and NA-specific antibody production103 during both primary13 and secondary immune responses.3 A possible mechanism for this phenomenon is that HA- or NA-specific B-cells capture influenza particles via surface Ig, and after internalization and processing, present to T cells the various processed protein components (e.g., M, NP) of the virus.103 Helper (CD4+) T cells are responsible for accelerating antibody responses following immunization.105 In humans, HA-specific T cells represent a major subset of proliferating T helper cells following influenza vaccination.106 Low frequencies of T cells directed to other influenza proteins can also be identified.107 Some non-responsiveness to conventional influenza vaccines may be linked to MHC haplotype, suggesting that T-cell responses are a critical component of protective immunity in humans.108 T helper response is also important to the control of influenza in mice.105 It has been shown that in mouse models, lifelong CD4 responses to influenza are maintained. In the mouse model, lifelong CD4 responses to influenza have been shown to be maintained.4,38,103,109 In humans, the study of CD4 durability is complicated by undocumented infections, but preliminary studies suggest that CD4 responses last at least several years post infection.96 The effect of CD4+ T cells and their contribution to priming the immune response is most evident when examining the effect of previous influenza exposure on the titer of antibody following re-vaccination. For example, the added benefit of a second dose (in terms of control of acute influenza) has long been apparent in young children never before vaccinated for or exposed to influenza.110 Studies of H1N1 vaccine in 1977 demonstrated that individuals born before 1957 (and who had been previously exposed to H1N1 viruses) required only a single dose of vaccine to achieve protective antibody titer levels, while those born after 1957 (i.e., never previously exposed to H1N1) required two doses.15 This difference suggests that T helper memory cells generated by infection pre-1957 may have been cross-reactive with the 1977 vaccine; this cross-reactivity may have helped to boost antibody titers to protective levels after a single immunization. These, and other analyses of historical data suggest that the protective impact of heterosubtypic immunity in humans may be due to conserved T-cell epitopes.111,112 Prospective studies evaluating the addition of T-cell epitopes to influenza vaccines are warranted. Memory T helper cells specific to a previous influenza strain can also assist with the development of qualitatively distinct heterotypic antibody responses.113 In addition, this relationship has

©

20

08

LA

ND

ES

BIO

SC

IEN

CE

.D

ON

Class I (CTL, CD8+) epitopes which are largely restricted by MHC haplotype,12 Class II (T helper) epitopes can be “promiscuous;” that is to say that a single sequence can fit MHC of various haplotypes.82 In both primary and secondary infection, whether homologous or heterologous, cellular immune responses are important in the host defense to influenza infection.19,59 Cognate help, one of the processes by which CD4 T cells assist the activation/maturation of B cells, was first characterized in influenza; however, little is known of the role of CD4+ TH cells as direct mediators of effector function.83 Influenza vaccine research over the past several decades has been strongly focused on generating B-cell responses; this bias is evident in the PubMed record. As of May 30, 2007, influenza vaccine publications referencing B cells outnumber those referencing T cells by more than three to one (4347:1435). The T-cell studies that have been done have focused primarily on CTL. Indeed, influenza vaccines are still designed without strong consideration for cognate T help; it thus follows that T-cell responses to the vaccine are rarely assessed. Below we summarize what is known about the role of T cells in influenza. We believe that the evidence supports the use of TH-cell epitopes to develop vaccines that generate better memory against B-cell epitopes. Cytotoxic T cells in influenza. Cytotoxic T cells (CTL) are so named because they can lyse infected cells that present antigenderived peptides in the context of MHC-antigen on their surface. Influenza-specific CTL can be either subtype-specific or reactive across subtypes.84 Early studies in mice showed that the majority of influenza-specific CTLs were reactive across subtypes.84 This high cross-reactivity is explained by the conserved antigenic targets of CTL. Only approximately 10–15% of the CTL precursor cells recognize HA85; unsurprisingly, there is little cross-reactivity between H1 and H3-HA.86 The primary CTL response in mice is dominated by two determinants: NP 366-37487 and PA 224-233.88 The murine CTLs also recognize an additional four sites: PB1703-711, NS2114-121, M1128-135 and PB1-F262-70.89 The secondary CTL response in mice is biased toward NP366-374, accounting for approximately 80% of the influenza-specific CD8+ T cells.90 The prevalence of anti-influenza CTLs correlates with the rate of viral clearance. Although CTL responses do not alter susceptibility to infection or subsequent infection,91 they do play a role in the containment of influenza once infection has occurred.17,92 T-cell responses following infection are known to wane over time.93 This may be in part a function of natural infection since native viral proteins NS1 and PB2 are known to inhibit the immune response.94 It is important to note that the CD4+ T helper cells enhance and amplify cytotoxic T cell (CTL) immune responses and have been shown to be important to fitness of CD8+ T-cell memory in influenza4 and to the longevity of CD8+ T cell responses in other viral models.95 How memory is maintained after acute infections is not yet known. T helper cells in influenza. T helper cells are so named because they are key mediators of diverse immune functions. Using conventional overlapping peptide methods, identifying conserved and promiscuous class II epitopes for influenza vaccine development has been near impossible due to virus strain diversity;117 only recently have confirmed sites been found.96 Immunizing humans and mice with whole influenza virus gives rise to populations of MHC-restricted www.landesbioscience.com

Human Vaccines

153


©

20

08

LA

ND

ES

BIO

SC

.

UT E

RIB

IEN

CE

.D

ON

The tools for development of conserved T help-enriched vaccines. The importance of T-helper cells in the defense against influenza has long been established; however, the goal of developing a Th cell-directed influenza vaccine has previously been complicated by (1) limited sequence information and (2) limitations in the bioinformatics tools available to analyze influenza sequences and to identify epitopes for genetically diverse populations. Today, thousands of influenza genome sequences are available thanks to the NIAID influenza genome project, making immunoinformatics—driven analysis of these genomes possible with robust algorithms. Addressing host diversity in MHC. As does the diversity of the virus, the diversity of human MHC (HLA) can also pose a challenge to vaccine development. EpiMatrix,118-121 an algorithm developed at Brown University and licensed to EpiVax, parses protein sequences into all its possible constituent 9-mer peptides and then estimates the probability of each segment’s ability to bind promiscuously to a panel of eight common and representative HLA Class II alleles. Together these eight archetype alleles cover the over 90% of human populations worldwide.122 The scores for each allele matrix are normalized so that they can be directly compared and regions of promiscuous epitopes easily identified. Addressing viral diversity in Th cell epitopes. “Clustered,” “superfamily” or “promiscuous” epitopes have a population coverage advantage over epitopes that are specific for one HLA allele. However, the problem of virus variability significantly complicates the development of promiscuous epitope-based vaccines. To address this problem, we developed EpiAssembler,123 another vaccine design computational tool. Among thousands of variant sequences, EpiAssembler identifies sets of overlapping, conserved and promiscuously immunogenic epitopes and assembles them into extended immunogenic consensus sequences (ICS). The resulting peptide is not a “pseudosequence” as such, since each constituent epitope occurs in its corresponding position in the native protein. In the case of HIV, for example, we used the ICS approach to design a peptidebased vaccine. While it is rare to find the full-length ICS peptide perfectly conserved in a given HIV isolate, the peptide nevertheless represents a significant percentage of circulating strains because every overlapping epitope is conserved in a large number (range 893 to 2,254) of individual HIV-1 strains. Of the 20 ICS peptides now tested, 19 provoked IFN-gamma release from HIV+ human PBMC as measured in ELISpot assays; the intensity of T cell response— ranging 200 to 500 spot-forming cells per peptide was substantial for immunocompromised HIV+ subjects.124 By focusing on conserved, MHC-promiscuous T-helper epitopes, the ICS approach has the potential to efficiently overcome the genetic variability of both virus and host.

IST

Future Directions for Influenza Vaccines

Practical application of cognate T cell help. We envision two possible approaches to take advantage of cognate T cell help to improve the efficacy, reactivity and duration of immune responses. The first would be to develop a peptide cocktail comprised of immunogenic consensus sequence (ICS) T-helper epitopes derived from a variety of HA and NA subtypes and from the highly conserved internal proteins. The second approach would be to develop composite antigens whose sequences are, in whole, or in part, optimized according to the ICS approach described above. While the peptide cocktail approach could be used as a broadbased prime for both conventional vaccines and infection, the second approach has the potential to replace the conventional vaccines. As described in the previous section, natural infection does prime cross-reactive TH cells and there is evidence to suggest that these cross-reactive T cells assist in hetero-subtypic immune responses in humans and in mice. Experimental evidence suggests that infection is insufficient to engender definitive immunity to subsequent infectious challenge.125,126 Reinfection occurs for two important reasons: first, the majority of the response to natural infection is raised to peptides that an individual human is unlikely to be exposed to again, and thus the full potential of the conserved antigenic content may not be realized. Second, any given influenza strain has limited conserved immunogenic content. In contrast, a single influenza vaccine could be designed to contain the conserved, promiscuous TH antigenic equivalent of multiple natural exposures or scores of conventional vaccines. A vaccine so designed may have the advantage of focusing the immune system a priori on those defenses most likely to be required long into the future. Immunogenic consensus sequence (ICS) peptide prime vaccine. How should one deliver a TH epitope-rich vaccine? Peptide cocktails have an excellent safety profile, and can be designed to maximize viral strain coverage. Disadvantages of peptide-based vaccines include expense of manufacture and validation. To address this, we developed the ICS approach which maximizes the antigenic coverage of each individual peptide. In addition, we recently developed Aggregatrix, an algorithm which iteratively searches for the minimum combination of peptides that achieves maximal cross-strain, cross-subtype representation.132 Several influenza researchers have explored conventional peptide immunization on a small scale. Although most such vaccines studied to date have been based on 1–5 epitopes and have been limited in terms of their sequence conservation, and/or hampered by MHCrestriction, results point to the promise of the approach.117,127-129 We believe that by increasing the number, quality and delivery of epitopes, and by boosting with whole HA and NA vaccines, it is possible to improve the efficacy of peptide epitope-based vaccines against influenza. Peptide-based vaccines also have the flexibility of antigenic breadth. One limitation of conventional vaccination, and of natural infection, is that the immune system focuses strongly on the most mutable immunogen of the virus—HA. Combining conserved Tcell epitopes from HA, and from other proteins, may circumvent this problem, boosting antibody response despite potential viral variability.13,14,102,103,130,131 In addition, broadening the T-cell repertoire might make it possible to impair viral escape and decrease viral loads sufficiently to disrupt transmission.

OT D

been observed with heterosubtypic pandemic strains: exposure to H9N2 induces protection against H5N1 challenge in chickens114 and mice.115,116 The viability of a CD4+ epitope-based vaccine seems to hold promise as Woodland et al.117 recently found that immunization with a single Th epitope provided a one-log reduction in viral titers early in infection. Broad reactivity and role in boosting B-cell response makes T-helper epitopes ideally suited for incorporation into influenza vaccines.

154

Human Vaccines



Dr. De Groot acknowledges that there is a potential conflict of interest related to her relationship with EpiVax and attests that the work contained in this research report is free of any bias that might be associated with the commercial goals of the company. References

OT D

IST

RIB

UT E

.

1. Osterholm MT. Preparing for the next pandemic. N Engl J Med 2005; 352:1839-42. 2. Anders EM, Peppard PM, Burns WH, White DO. In vitro antibody response to influenza virus. T cell dependence of secondary response to hemagglutinin. J Immunol 1979; 123:1356-61. 3. Johansson BE, Moran TM, Bona CA, Kilbourne ED. Immunologic response to influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin: Reduced generation of neuramindase specific Helper T cells in hemagglutinin primed mice. J Immunol 1987; 139: 2015-9. 4. Johansen P, Stamou P, Tascon RE, Lowrie DB, Stockinger B. CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells. Eur J Immunol 2004; 34:91-7. 5. Kilbourne ED. What are the prospects for a universal influenza vaccine? Nat Med 1999; 5:1119-20. 6. Choppin P, and Tamm I. Studies of two kinds of virus particles which comprise influenza A2 virus strains. Reactivity with virus inhibitors in normal sera. J Exp Med 1960; 112:921. 7. Johansson B, Bucher D, and Kilbourne E. Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection. J Virology 1989; 63:1239. 8. Compans R, Klenk H-D, Caliguiri L, Choppin P. Influenza virus proteins. Analysis of polypeptides of the virion and identification of spike glycoproteins. Virology 1970; 42:880. 9. Kilbourne ED. Comparative efficacy of neuraminidase-specific and conventional influenza virus vaccines in induction of antibody to neuraminidase in humans. J Infect Dis, 1976; 134: 385. 10. Murphy BR, Nelson DL, Wright PF, Tierney EL, Phelan MA, Chanock RM. Secretory and systemic immunological response in children infected with live attenuated influenza A virus vaccines. Infect Immun 1982; 36:1102-8. 11. Burlington DB, Clements ML, Meiklejohn G, Phelan M, Murphy BR. Hemagglutininspecific antibody responses in immunoglobulin G, A, and M isotypes as measured by enzyme-linked immunosorbent assay after primary or secondary infection of humans with influenza A virus. Infect Immun 1983; 41: 540-5 12. Paul WE and Benacerraf B. Functional specificity of thymus-dependent lymphocytes. Science 1977; 195:1293 13. Scherle PA, Gerhard W. Functional analysis of influenza-specific helper T cell clones in vivo. T cells specific for internal viral proteins provide cognate help for B cell responses to hemagglutinin. J Exp Med 1986; 164:1114-28. 14. Russell SM, Liew FY. T cells primed by influenza virion internal components can cooperate in the antibody response to haemagglutinin. Nature 1979; 280:147-8. 15. LaMontagne JR, Noble GR, Quinnan GV, Curlin GT, Blackwelder WC, Smith JI, Ennis FA, Bozeman FM. Summary of clinical trials of inactivated influenza vaccine 1978. Rev Infect Dis 1983; 5:723-36. 16. Liu W, Li H, Chen Y-H. N-terminus of M2 protein could induce antibodies with inhibitory activity against influenza virus replication. FEMS Immunol Med Microbiol 2003; 35:141-6. 17. Flynn KJ, Belz GT, Altman JD, Ahmed R, Woodland DL, Doherty PC. Virus-specific CD8+ T cells in primary and secondary influenza pneumonia. Immunity 1998; 8:683-91. 18. Johansson BE, Bucher DJ, Pokorny BA and Kilbourne ED. Identification of PR-8 M1 protein influenza high-yield reassortants by M1-specific monoclonal antibodies. Virology 1989; 171:634-6. 19. Doherty PC, Turner SJ, Webby RG, Thomas PG. Influenza and the challenge for immunology. Nat Immunol 2006; 7:449-55. 20. Treanor JJ, Schiff GM, Couch RB, Cate TR, Brady RC, C. Hay M, Wolff M, She D, Cox MJM. Dose-Related Safety and Immunogenicity of a Trivalent Baculovirus-Expressed Influenza-Virus. J Inf Dis 2006; 193:1223. 21. Powers D, Smith G, Anderson E, Kennedy D, Hackett C, Wilkinson B, et al. Evaluation of a recombinant hemagglutinin expressed in insect cells as an influenza vaccine in young and elderly adults. J Infect Dis 1996; 173:1467-70. 22. Kilbourne ED, Johansson BE, Moran TM, Wu S, Pokorny BA, Xu X, and Cox N. Implications for vaccine production of influenza A virus hemagglutinin polymorphism. Pleotropic antigenic variant of A/Shangai/11/87 (H3N2) virus selected as high-yield reassortant. J Gen Virol 1993; 74:1311-6. 23. Huddleston JA, Brownlee GG. The sequence of the nucleoprotein gene of human influenza A virus, strain A/NT/60/68. Nucleic Acids Res 1982; 10:1029-38. 24. Berton MT, Naeve CW, Webster RG. Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies. J Virol 1984; 52: 919-27. 25. Air GM, Laver WG, Webster RG. Antigenic variation in influenza viruses. Contrib Microbiol Immunol 1987; 8:20-59. Review. 26. Cruse JM, Lewis RB. Contemporary concepts of antigenic variation. Contra Micro Immuno 1987; 8:1-19.

.D

ON

ICS protein based vaccines. By extending the approach described above, it is possible to develop complete synthetic antigens whose sequences are optimized for T-helper potential. With an eye to structural considerations, even HA-only vaccines could be optimized in this way, enabling primary cognate T help to be maximized and B-cell memory to be elicited. We believe that the ideal vaccine would include whole HA and NA in addition to internal proteins; some or all of these antigens could be optimized using the ICS approach. Physically linking the HA and NA to the internal proteins (eg. assembled as a VLP), would further maximize primary cognate T help, since B cells that capture HA or NA would be able to process and present T-helper epitopes derived from more and various proteins. Such a vaccine would likely be able to stimulate potent, relevant and long-lasting immune responses while being safe for use in those at highest risk for influenza. Unlike a conventional adjuvant, the cognate T-helper epitopes would do more than simply augment immune responses to the vaccine; they would also assist in boosting immune responses to related influenza strains. Using EpiAssembler, we have designed several ICS sequences for influenza. As compared with immunogenic consensus sequences, randomly-selected counterparts, on average, contain half as many binding motifs and cover a third fewer isolates. To develop vaccines of equivalent antigenic “payload” using conventional methods would be prohibitively expensive as it would require including multiple different variants of each antigen. While the influenza ICS sequences, and ICS proteins have not yet been tested, we believe that these and similar approaches that harness conserved T help deserve consideration.

CE

Summary

©

20

08

LA

ND

ES

BIO

SC

IEN

In summary, new approaches to influenza vaccines are needed. Humans possess little or no preexisting immunity to emerging antigenically drifted and shifted influenza variants, each with the potential to cause epidemics or pandemics. Since 1995, several novel avian subtypes have crossed the species barrier to humans and have caused a spectrum of mild to severe and even fatal human disease —H5N1, H7N7, H9N2, H7N3 and H10N7. Through adaptive mutation or reassortment, H5N1 or other avian subtypes have the potential to evolve to achieve highly pathogenic human-to-human transmission. Additionally, seasonal influenza can evade immune surveillance as a result of vaccine failure and can emerge as an epidemic strain. The development of safe and effective vaccines against influenza, both seasonal and pandemic, is an urgent and achievable public health priority. The importance of cognate T help was established decades ago; new tools and technologies have made it possible— perhaps imperative—to harness that helper potential to develop potent and broad-spectrum vaccines against influenza. Acknowledgements

We thank Lenny Moise for invaluable assistance in the editing of this manuscript. Conflict of interest

One of the contributing authors, Anne S. De Groot, is CEO and majority shareholder at EpiVax, Inc., a privately owned vaccine design company located in Providence, RI. Dr. De Groot is also a faculty member at Brown University. www.landesbioscience.com

Human Vaccines

155


OT D

IST

RIB

UT E

.

56. Johansson BE, Brett IC, Focosi D, Poland GA. An Inactivated Subvirion Influenza A (H5N1) Vaccine. NEJM 2006; 354:2724-5. 57. Black RA, Rota PA, Gorodkova N, Klenk HD, Kendal AP. Antibody response to M2 protein of influenza A virus expressed in insect cells. J Gen Virol 1993; 74:143-6. 58. Mozdzanowska K, Feng J, Eid M, Kragol G, Cudic M, Otvos L Jr, Gerhard W. Induction of influenza type A virus-specific resistance by immunization of mice with a synthetic multiple antigenic peptide vaccine that contains ectodomains of matrix protein 2. Vaccine 2003; 21:2616-26. 59. Dong L, Mori I, Hossain MJ, Kimura Y. The senescence-accelerated mouse shows agingrelated defects in cellular but not humoral immunity against influenza virus infection. J Infect Dis 2000; 182:391-6. 60. Boon AC, de Mutsert G, van Baarle D, et al. Recognition of homo- and heterosubtypic variants of influenza A viruses by human CD8+ T lymphocytes. J Immunol 2004; 172:2453-60. 61. Kreijtz JH, Bodewes R, van Amerongen G, Kuiken T, Fouchier RA, Osterhaus AD, Rimmelzwaan GF. Primary influenza A virus infection induces cross-protective immunity against a lethal infection with a heterosubtypic virus strain in mice. Vaccine 2007; 25:612-20. 62. Kamperschroer C, Dibble JP, Meents DL, Schwartzberg PL, Swain SL. SAP is required for Th cell function and for immunity to influenza. J Immunol 2006; 177:5317-27. 63. Belz GT, Wodarz D, Diaz G, Nowak MA, Doherty PC. Compromised influenza virus-specific CD8(+)-T-cell memory in CD4(+)-T-cell deficient mice. J Virol 2002; 76:12388-93. 64. Cardin RD, Brooks JW, Sarawar SR, Doherty PC. Progressive loss of CD8+ T cell-mediated control of a gamma-herpesvirus in the absence of CD4+ T cells. J Exp Med 1996; 184:863-71. 65. Brooks JW, Hamilton-Easton AM, Christensen JP, Cardin RD, Hardy CL, Doherty PC. Requirement for CD40 ligand, CD4(+) T cells, and B cells in an infectious mononucleosislike syndrome. J Virol 1999; 73:9650-4. 66. Johansson BE, Pokorny BA, Tiso VA. Supplementation of conventional trivalent influenza vaccine with purified viral N1 and N2 neuraminidases induces a balanced immune response without antigenic competition. Vaccine 2002; 20:1670-4. 67. Kendal AP, Bozeman FM, Ennis FA. Further studies of the neuraminidase content of inactivated influenza vaccines and the neuraminidase antibody responses after vaccination of immunologically primed and unprimed populations. Infect Immun 1980; 29:966-71. 68. Nestorowicz A, Laver G, Jackson DC. Antigenic determinants of influenza virus haemagglutinin. X. A comparison of the physical and antigenic properties of monomeric and trimeric forms. J Gen Virol 1985; 66:1687-95. 69. Muller, GM., Shapira, M and Arnon, R. Anti-Influenza Response Achieved by Immunization with a synthetic conjugate. PNAS 1982; 79:569-73 70. Nestorowicz A, Tregear GW, Southwell CN, Martyn J, Murray JM, White DO, Jackson DC. Antibodies elicited by influenza virus hemagglutinin fail to bind to synthetic peptides representing putative antigenic sites. Mol Immunol 1985; 22:145-54. 71. Green N, Alexander H, Olson A, Alexander S, Shinnick TM, Sutcliffe JG, Lerner RA. Immunogenic structure of the influenza virus hemagglutinin. Cell 1982; 28:477-87. 72. Russ G, Styk B, Polakova K. Antigenic glycopeptides HA1 and HA2 of influenza virus hemagglutinin. II. Reactivity with rabbit sera against intact virus and purified undissociated hemagglutinin. Acta Virol 1978; 22:371-82. 73. Graves PN, Schulman JL, Young JF, Palese P. Preparation of influenza virus subviral particles lacking the HA1 subunit of hemagglutinin: unmasking of cross-reactive HA2 determinants. Virology 1983; 126:106-16 74. Varghese JN, Laver, WG and Colman PM. Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature 1983; 303:35-40. 75. Colman PM, Varghese JN, Laver WG. Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature 1983; 303:41-44. 76. Biddison WE, Doherty PC, Webster RG. Antibody to influenza virus matrix protein detects a common antigen on the surface of cells infected with type A influenza viruses. J Exp Med 1977; 146:690-7. 77. Yoden S, Kida H, Kuwabara M, Yanagawa R, Webster RG. Spin-labeling of influenza virus hemagglutinin permits analysis of the conformational change at low pH and its inhibition by antibody. Virus Res 1986; 3:251-61. 78. Kilbourne ED, Palese P, Schulman JL. Inhibition of viral neuraminidase as a new approach to the prevention of influenza. In Perspectives in virology Vol 9 (Ed. Pollard, M.) New York, Academic Press, 1975; 99-113. 79. van Wyke KL, Hinshaw VS, Bean WJ Jr, Webster RG. Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies. J Virol 1980; 35:24-30. 80. Johansson BE, Kilbourne ED. Dissociation of influenza virus neuraminidase from matrix and nulceoprotein eliminates cognate help and antigenic competition. Virology 1996; 225:136-44. 81. Lawson C, Bennink J, Restifo N, Yewdell J, Murphy BR. Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge. 1994; J Virol 68:3505. 82. Caro-Aguilar I, Rodríguez A, Calvo-Calle JM, Guzmán F, De la Vega P, Patarroyo ME, Galinski MR, Moreno A. Plasmodium vivax Promiscuous T-Helper Epitopes Defined and Evaluated as Linear Peptide Chimera Immunogens Infect Immun 2002; 70: 3479-92. 83. Thomas PG, Keating R, Hulse-Post DJ, Doherty PC. Cell-mediated protection in influenza infection. Emerg Infect Dis 2006; 12:15-22.

©

20

08

LA

ND

ES

BIO

SC

IEN

CE

.D

ON

27. Campbell CH, Webster RG, and Breese SS Jr. The in vivo production of “new” influenza A viruses. Virology 1971; 44:317-28. 28. Taubenberger JK, Reid AH, Lourens RM, Wang R, Jin G, Fanning TG. Characterization of the 1918 influenza virus polymerase genes. Nature 2005; 437:889–93. 29. Henry M, Kieny M, Thompson, R. Immediate and sustained action required to sharply increase pandemic influenza vaccine supply. WHO mediacentre. Last updated 23 October 2006. Retrieved 15 November, 2006 from http://www.who.int/mediacentre/news/releases/2006/pr58/en/index.html. 30. Brett IC, Johansson BE. Immunization against influenza A virus: comparison of conventional inactivated, live-attenuated and recombinant baculovirus produced purified hemagglutinin and neuraminidase vaccines in a murine model system. Virology 2005; 339:273-80. 31. Cox RJ, Brokstad KA, Ogra P. Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines. Scand J Immunol 2004; 59:1-15. 32. Glezen WP. Cold-adapted, live attenuated influenza vaccine. Expert Rev Vaccines 2004; 3:131-9. 33. Gross P, Ennis F, Gaerlan L, Denson L, Denning C, Schiffman D. A controlled doubleblind comparison of the reactogenicity, immunogenicity, and protective efficacy of wholevirus and split-product influenza vaccines in children. J Inf Dis 1977; 136:623. 34. Rivetti D, Jefferson T, Thomas R, Rudin M, Rivetti A, Di Pietrantonj C, Demicheli V. Vaccines for preventing influenza in the elderly. Cochrane Database Syst Rev 2006; 3: CD004876. 35. Besselaar TG, Botha L, McAnerney JM, Schoub BD. Antigenic and molecular analysis of influenza A (H3N2) virus strains isolated from a localised influenza outbreak in South Africa in 2003. J Med Virol 2004; 73:71-8. 36. McLaren C, Verbonitz MW, Daniel S, Guggs G, Ennis F. Effect of priming infection on serologic response to whole and subunit influenza virus vaccines in animals. J Immuno 1977; 125:2679 37. Wise T, Polin R, Mazur M, Ennis F. Serologic responses after two sequential doses of influenza A/New Jersey/76 virus vaccine in normal young adults. J Inf Dis 1977;136:5496. 38. Johansson BE, Moran TM, Kilbourne ED. Antigen-presenting B cells and helper T cells cooperatively mediate intravirionic antigenic competition between influenza A virus surface glycoproteins. PNAS USA 1987; 84:6869-73. 39. Rasmussen IB, Lunde E, Michaelsen TE, Bogen B, Sandlie I. The principle of delivery of T cell epitopes to antigen-presenting cells applied to peptides from influenza virus, ovalbumin, and hen egg lysozyme: implications for peptide vaccination. Proc Natl Acad Sci USA 2001; 98:10296-301. 40. Lanzavecchia A. Antigen-specific interaction between T and B cells. Nature 1985; 314: 537. 41. Belshe RB. A review of attenuation of influenza viruses by genetic manipulation. Am J Respir Crit Care Med 1995; 152:S72-5. 42. McMicheal A, Grotch F, Cullen P, Askonas B, and Webster R. The human cytotoxic T cell response to influenza vaccination. Clin Exp Immunol 1981; 43:276. 43. McLaren C, Verbonitz MW, Daniel S, Guggs G, Ennis F. Effect of priming infection on serologic response to whole and subunit influenza virus vaccines in animals. J Immuno 1977; 125:2679 44. Belshe R, Gruber W, Mendelman P, Cho I, Reisinger K, Block S, et al. Efficacy of vaccination with live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine against a variant (A/Sydney) not contained in the vaccine. J Pediatrics 2000; 136:168-175. 45. Kilbourne E. Comparative efficacy of neuraminidase-specific and conventional influenza virus vaccines in the induction of anti-neuraminidase antibody in man. J Inf Dis 1976; 134:384. 46. Belshe RB, Edwards KM, Vesikari T, Black SV, Walker RE, Hultquist M, Kemble G, Connor EM; CAIV-T Comparative Efficacy Study Group. Live attenuated versus inactivated influenza vaccine in infants and young children. N Engl J Med 2007; 356:685-96 47. Treanor JJ, Wilkinson BE, Masseoud F, Hu-Primmer J, Battaglia R, O’Brien D, Wolff M, Rabinovich G, Blackwelder W, Katz JM. Safety and immunogenicity of a recombinant hemagglutinin vaccine for H5 influenza in humans. Vaccine 2001;19:1732-7. 48. Treanor JJ, Schiff GM, Hayden FG, Brady RC, Hay CM, Meyer AL, Holden-Wiltse J, Liang H, Gilbert A, Cox M. Safety and Immunogenicity of a Baculovirus-Expressed Hemagglutinin Influenza Vaccine A Randomized Controlled Trial. JAMA 2007; 297:1577-82. 49. Epstein SL. Control of influenza virus infection by immunity to conserved viral features. Expert Rev Anti Infect Ther 2003; 1:627-38. 50. Cassetti MC, Couch R, Wood J, Pervikov Y. Report of meeting on the development of influenza vaccines with broad spectrum and long-lasting immune responses, World Health Organization, Geneva, Switzerland, 26-27 February 2004. Vaccine 2005; 23:1529-33. 51. Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M. Safety and immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. N Engl J Med 2006; 354:1343-51. 52. Nicholson KG, Colegate AE, Podda A, et al. Safety and antigenicity of non-adjuvanted and MF-59 adjuvanted/Duck/Singapore/97 (H5N3) vaccine: a randomized trial of two potential vaccines against H5N1 influenza. Lancet 2001; 357:1937-43. 53. Poland GA. Vaccines against avian influenza-a race against time. N Engl J Med 2006; 354:1411-3. 54. Wood JM. Developing vaccines against pandemic influenza. Philos Trans R Soc Lond B Biol Sci 2001; 356:1953-60. 55. Hehme N, Engelmann H, Ku¨nzel W, Neumeier E, Sanger R. Pandemic preparedness: lessons learnt from H2N2 and H9N2 candidate vaccines. Med Microbiol Immunol (Berl) 2002; 191:203-8.

156

Human Vaccines



OT D

IST

RIB

UT E

.

112. Sonoguchi T, Naito H, Hara M, Takeuchi Y, Fukumi H. Cross-subtype protection in humans during sequential overlapping and/or concurrent epidemics caused by H3N2 and H1N1 influenza viruses. J Infect Dis 1985; 151:81-8. 113. Marshall D, Sealy R, Sangster M, Coleclough C. TH Cells Primed During Influenza Virus Infection Provide Help for Qualitatively Distinct Antibody Responses to Subsequent Immunization1. The Journal of Immunology 1999; 163:4673-4682. 114. Seo SH, Webster RG. Cross-reactive, cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in Hong Kong poultry markets. J Virol 2001; 75:2516-25. 115. O’Neill E, Krauss SL, Riberdy JM, Webster RG, Woodland DL. Heterologous protection against lethal A/HongKong/156/97 (H5N1) influenza virus infection in C57BL/6 mice. J Gen Virol 2000; 81:2689-96 116. Anders EM, Katz JM, Jackson DC, White DO. In vitro antibody response to influenza virus. Specificity of helper T cell recognizing hemagglutinin. J Immunol 1981; 127:669-72. 117. Crowe SR, Miller SC, Brown DM, Adams PS, Dutton RW, Harmsen AG, Lund FE, Randall TD, Swain SL, Woodland DL. Uneven distribution of MHC class II epitopes within the influenza virus. Vaccine 2006; 24:457-67. 118. De Groot AS, Jesdale BM, Szu E, Schafer JR. An interactive web site providing MHC ligand predictions: application to HIV research. AIDS Res and Hum Retroviruses 1997; 13:539-41. 119. Schafer JA, Jesdale BM, George JA, Kouttab NM, De Groot AS. Prediction of well-conserved HIV-1 ligands using a Matrix-based Algorithm, EpiMatrix. Vaccine 1998; 16:1880-4. 120. De Groot AS, Bosma A, Chinai N, Frost J, Jesdale B, Gonzalez M, Martin W, Saint-Aubin C. From genome to vaccine: In silico predictions, ex vivo verification. Vaccine 2001; 19:4385-95. 121. De Groot AS, Sbai H, Jesdale B, Martin W, Saint Aubin C, Bosma A, Lieberman J, Skowron GA, Mansourati F, Mayer K. Mapping Cross-clade HIV-1 Vaccine Epitopes Using a Bioinformatics Approach. Vaccine 2003; 21:4486-504. 122. Southwood S, Sidney J, Kondo A, del Guercio MF, Appella E, Hoffman S, Kubo RT, Chesnut RW, Grey HM, Sette A. Several common HLA-DR types share largely overlapping peptide binding repertoires. J Immunol 1998; 160:3363-73. 123. De Groot AS, Bishop E, Khan B, Lally M, Marcon L, Franco J, Mayer K, Carpenter C, Martin W. Engineering immunogenic consensus T helper epitopes for a cross-clade HIV vaccine. Methods 2004; 34:476-87. 124. De Groot AS, Marcon L, Bishop EA, Rivera D, Kutzler M, Weiner DB, Martin W. HIV vaccine development by computer assisted design: the GAIA vaccine. Vaccine 2005; 23:2136-48. 125. Fox JP, Hall CE. Viruses in families. Littleton, MA: PSG Publishing Company, 1980. 126. Frank AL, Taber LH, Glezen WP, Paredes A, Couch RB. Reinfection with influenza A (H3N2) virus in young children and their families. J Inf Disease 1979; 140: 829-36. 127. Levi R, Arnon R. Synthetic recombinant influenza vaccine induces efficient long-term immunity and cross-strain protection. Vaccine 1996; 14:85-92. 128. Jeon SH, Ben-Yedidia T, Arnon R. Intranasal immunization with synthetic recombinant vaccine containing multiple epitopes of influenza virus. Vaccine 2002; 20:2772-80. 129. Mack J, Falk K, Rotzschke O, Walk T, Strominger JL, Jung G. Synthesis of linear and comblike peptide constructs containing up to four copies of a T cell epitope and their capacity to stimulate T cells. J Pept Sci 2001; 7:338-45. 130. Santra S, Barouch DH, Kuroda MJ, Schmitz JE, Krivulka GR, Beaudry K, Lord CI, Lifton MA, Wyatt LS, Moss B, Hirsch VM, Letvin NL. Prior vaccination increases the epitopic breadth of the cytotoxic T-lymphocyte response that evolves in rhesus monkeys following a simian-human immunodeficiency virus infection. J Virol 2002; 76:6376-81. 131. Subbramanian RA, Kuroda MJ, Charini WA, Barouch DH, Costantino C, Santra S, Schmitz JE, Martin KL, Lifton MA, Gorgone DA, Shiver JW, Letvin NL. Magnitude and diversity of cytotoxic-T-lymphocyte responses elicited by multiepitope DNA vaccination in rhesus monkeys. J Virol 2003; 77:10113-8. 132. Rivera DS, McMurry JA, Buus S, Martin W, De Groot AS. Identification of immunogenic HLA-B7 “Achilles’ heel” epitopes within highly conserved regions of HIV. Vaccine 2007; In Press.

©

20

08

LA

ND

ES

BIO

SC

IEN

CE

.D

ON

84. Braciale TJ. Immunologic recognition of influenza virus-infected cells. I. Generation of a virus-strain specific and a cross-reactive subpopulation of cytotoxic T cells in the response to type A influenza viruses of different subtypes. Cell Immunol 1977; 33:423-36. 85. Andrew ME, Coupar BE, Ada GL, Boyle DB. Cell-mediated immune responses to influenza virus antigens expressed by vaccinia virus recombinants. Microb Pathog 1986; 1:443-52. 86. Braciale TJ, Andrew ME, Braciale VL. Heterogeneity and specificity of cloned lines of influenza-virus specific cytotoxic T lymphocytes. J Exp Med 1981; 153:910-23 87. Townsend, ARM, Rothbard J. Gotch FM, Bahadur G, Wraith D, and McMichal AJ. The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 1986; 44:959-68. 88. Riberdy JM, Christensen JP, Branum K, Doherty PC. Diminished primary and secondary influenza virus-specific CD8(+) T-cell responses in CD4-depleted Ig(-/-) mice. J Virol 2000; 74:9762-5. 89. Chen W, Calvo PA, Malide D, Gibbs J, Schubert U, Bacik I, et al. A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 2001; 7:1306-12. 90. Marshall DR, Turner SJ, Belz GT, Wingo S, Andreansky S, Sangster MY, et al. Measuring the diaspora for virus-specific CD8+ T cells. PNAS USA 2001; 98:6313-8. 91. McMicheal A, Grotch F, Cullen P, Askonas B, and Webster R. The human cytotoxic T cell response to influenza vaccination. Clin Exp Immunol 1981; 43:276. 92. Plotnicky H, Cyblat-Chanal D, Aubry JP, Derouet F, Klinguer-Hamour C, Beck A, Bonnefoy JY, Corvaia N. The immunodominant influenza matrix T cell epitope recognized in human induces influenza protection in HLA-A2/K(b) transgenic mice. Virology 2003; 309:320-9. 93. Powell TJ, Brown DM, Hollenbaugh JA, Charbonneau T, Kemp RA, Swain SL, Dutton RW. CD8+ T cells responding to influenza infection reach and persist at higher numbers than CD4+ T cells independently of precursor frequency. Clin Immunol 2004; 113:89-100. 94. Neuman G, Kawaoka Y. Host range restriction and pathogenicity in the context of influenza pandemic. Emerging Infectious Diseases 2006; 12: 881-6. 95. Walter E A, Greenberg PD, Gilbert MJ, Finch RJ, Watanabe KS, Thomas ED, Riddell SR. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med 1995; 333: 1038-44. 96. Gelder CM, Welsh KI, Faith A, Lamb JR, Askonas BA. Human CD4+ T-cell repertoire of responses to influenza A virus hemagglutinin after recent natural infection. J Virol 1995; 69:7497-506. 97. Hackett CJ, Hurwitz JL, Dietzschold B, Gerhard W. A synthetic decapeptide of influenza virus hemagglutinin elicits helper T cells with the same fine recognition specificities as occur in response to whole virus. J Immunol 1985; 135:1391-4. 98. Lamb JR, Eckels DD, Phelan M, Lake P, Woody JN. Antigen-specific human T lymphocyte clones: viral antigen specificity of influenza virus-immune clones. J Immunol 1982; 128:1428-32 99. Hurwitz JL, Hackett CJ, McAndrew EC, Gerhard W. Murine TH response to influenza virus: recognition of hemagglutinin, neuraminidase, matrix, and nucleoproteins. J Immunol 1985; 134:1994-8. 100. Katz JM, Laver WG, White DO, and Anders, EM. Recognition of influenza virus hemagglutinin by subtype-specific and cross-reactive proliferative T cells: contribution of HA1 and HA2 polypeptide chains. J Immunol 1985; 134:616-22. 101. Hurwitz JL, Herber-Katz E, Hackett CJ, Gerhard W. Characterization of the murine TH response to influenza virus hemagglutinin: evidence for three major specificities. J Immunol 1984; 133:3371-7. 102. Scherle PA, Gerhard W. Differential ability of B cells specific for external vs. internal influenza virus proteins to respond to help from influenza virus-specific T-cell clones in vivo. PNAS USA 1988; 85:4446-50. 103. Johansson BE, Moran TM, Kilbourne ED. Antigen-presenting B cells and helper T cells cooperatively mediate intravirionic antigenic competition between influenza A virus surface glycoproteins. PNAS USA 1987; 84:6869-73. 104. Virelizier JL, Postlethwaite R, Schild GC, Allison AC. Antibody responses to antigenic determinants of influenza virus hemagglutinin. Thymus dependence of antibody formation and thymus independence of immunological memory. J Exp Med 1974; 140:1559-70. 105. Schneider C, Van Regenmortel MH. Immunogenicity of free synthetic peptides corresponding to T helper epitopes of the influenza HA 1 subunit. Induction of virus cross reacting CD4+ T lymphocytes in mice. Arch Virol 1992; 125:103-19. 106. Novak EJ, Liu AW, Nepom GT, Kwok WW. MHC class II tetramers identify peptide-specific human CD4(+) T cells proliferating in response to influenza A antigen. J Clin Invest 1999; 104:R63-7. 107. Danke NA, Kwok WW. HLA class II-restricted CD4+ T cell responses directed against influenzaviral antigens postinfluenza vaccination. J Immunol 2003; 171:3163-9. 108. Gelder CM, Lambkin R, Hart KW, Fleming D, Williams OM, Bunce M, Welsh KI, Marshall SE, Oxford J. Associations between human leukocyte antigens and nonresponsiveness to influenza vaccine. J Infect Dis 2002; 185:114-7. 109. Doherty PC, Turner SJ, Webby RG, Thomas PG. Influenza and the challenge for immunology. Nat Immunol 2006; 7:449-55. Review. 110. Quilligan JJ, Francis T. Serological Response to Intranasal Administration of Inactive Influenza Virus in Children. J Clin Invest 1947; 26:1079-87. 111. Epstein SL. Prior H1N1 influenza infection and susceptibility of Cleveland Family Study participants during the H2N2 pandemic of 1957: an experiment of nature. J Infect Dis 2006; 193:49-53.

www.landesbioscience.com

Human Vaccines

157