流感疫苗的未来发展------2010 NEJM

review article T h e n e w e ngl a nd j o u r na l o f m e dic i n e n engl j med 363;21 nejm.org november 18, 20102036 Current Concepts Influenza Vaccines for the Future Linda C. Lambert, Ph.D., and Anthony S. Fauci, M.D. From the Division of Microbiology and Infectious Diseases (L.C.L.) and the Of- fice of the Director (A.S.F.), National In- stitute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. Address reprint requests to Dr. Lam- bert at the Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, 6610 Rockledge Dr., Bethesda, MD 20892, or at lclambert@mail.nih.gov. N Engl J Med 2010;363:2036-44. Copyright © 2010 Massachusetts Medical Society. Each year, seasonal epidemics of influenza cause serious illness and death throughout the world. In the United States, the annual burden of disease is estimated to be 25 million to 50 million cases of influenza, resulting in an average of 225,000 hospitalizations. Over the past three decades, the estimated number of influenza-associated deaths per year in the United States has ranged from 3349 to 48,614. The majority of deaths (>90%) occur among elderly persons, usually those with chronic underlying health conditions.1-3 The World Health Orga- nization uses these estimates to extrapolate a likely global disease burden from influenza of up to 1 billion infections, 3 million to 5 million cases of severe disease, and between 300,000 and 500,000 deaths annually.1 Pandemics of influenza with varying rates of illness and death have occurred throughout history; the most no- table was the 1918–1919 pandemic, which claimed an estimated 50 million to 100 million lives worldwide.4 First isolated from humans in 1933,5 influenza viruses contain 8 single-stranded RNA segments encoding 11 proteins (Fig. 1). There are three types of influenza viruses: A, B, and C, with types A and B causing annual human epidemics. A key feature of the influenza virus is its error-prone polymerase, which results in an accumulation of genetic mutations that are selected for in hemagglutinin (HA) and to a lesser extent neuraminidase (NA) — the major surface glycoproteins of the virus. This antigenic drift of the HA protein renews our susceptibility to influenza viruses and is the basis for frequent updating of the composition of seasonal influ- enza vaccines. Protection after natural infection is primarily mediated by HA-specific antibodies in serum and mucosa, with the presence of antibodies against NA, con- served influenza proteins, and T-cell responses correlating with reduced disease severity. A novel virus can emerge in humans either through direct interspecies trans- mission or as a result of molecular exchanges between influenza viruses that al- ready infect humans. Because the influenza virus genome is segmented, coinfection of a single host cell with two or more different influenza viruses can result in a reassortment (or shuffle) of their genetic material. The antigenic shift can lead to a pandemic if the resulting progeny virus contains an HA protein to which humans have no preexisting immunity, if it has an efficient replication-competent set of in- ternal genes, and if it can readily spread from human to human — as was the case with the 2009 H1N1 virus. Vaccines for Influenz a Con trol Vaccination is the primary strategy for the prevention and control of influenza.8,9 Although both inactivated vaccines and the live attenuated vaccine are effective in preventing influenza and its associated complications, the protection they confer varies widely, depending on the antigenic match between the viruses in the vaccine The New England Journal of Medicine Downloaded from nejm.org by liang qiao on December 16, 2010. For personal use only. No other uses without permission. Copyright © 2010 Massachusetts Medical Society. All rights reserved. current concepts n engl j med 363;21 nejm.org november 18, 2010 2037 and those that are circulating during a given in- fluenza season and on the recipient’s age and health status.10 More effective vaccination options are needed, especially for persons who have a re- duced immunologic response to vaccination, in- cluding the elderly and those with chronic under- lying disease. A step toward this goal is the recently approved high-dose, inactivated influ- enza vaccine.11 Seasonal influenza vaccines are trivalent. Each dose is formulated to contain three viruses (or their HA proteins) representing the influenza A H3N2, influenza A H1N1, and influenza B strains considered to be the most likely to circulate in the upcoming influenza season. The strains for Northern Hemisphere vaccines are generally se- lected in February for use in the following season. Inactivated-vaccine production begins with the generation of vaccine reference strains — hybrid viruses with the HA and NA genes from the drifted variant combined with other genes from a laboratory strain adapted to grow well in eggs. This process can take several weeks or longer.12,13 Manufacturers sometimes find that the new strain still grows poorly in eggs or yields low levels of HA protein and needs to be further “egg-adapted” through serial passage. In contrast, plasmid-based reverse-genetics technology is now being used to reliably generate reference strains within a short- er time frame and to improve their growth in eggs.14,15 Traditionally, from February to late sum- mer, manufacturers amplify the vaccine viruses in hundreds of millions of embryonated chicken eggs and inactivate or purify them. These vac- cines are then formulated, packaged, and distrib- uted beginning early in the fall for administration before the peak of the influenza season, which usually occurs after December. Ch a llenges t o Producing H1N1 Vaccine When human 2009 H1N1 viruses were identified in the spring of 2009, vaccine manufacturers were well into their annual production of seasonal in- fluenza vaccine for the 2009–2010 season.16 Ow- ing to the uncertainty of the evolving outbreak, a decision was made to continue the seasonal vac- cine production and to begin separate production of a vaccine against the new virus. The persistence and dominance of the 2009 H1N1 virus became evident throughout the summer, and the number of cases of 2009 H1N1 virus–related influenza increased in August and September, compressing the vaccine production timeline further, by sev- eral months. An additional challenge for the in- activated-vaccine manufacturers was the substan- tially lower-than-expected yields of HA protein, resulting in fewer doses being available initially. For the immediate future, priorities have been established for overcoming the rate-limiting steps in the production of inactivated vaccines. These include wider implementation of technologies, such as reverse genetics, to generate vaccine ref- erence strains optimized to grow well in eggs, and new methods to accelerate vaccine potency and sterility testing, which would substantially shorten the time from strain selection to release of vaccine.17 The live attenuated 2009 H1N1 viruses reached very high titers in eggs, allowing this vaccine to be the first one distributed. However, several barriers need to be overcome for broader use of the live at- tenuated vaccine in a future pandemic, including its approval for use in age groups other than those for which it is currently indicated (i.e., only healthy persons 2 to 49 years of age) and the development of formulations that can be administered without a special nasal-spray device (i.e., nose drops). For both inactivated and live attenuated vaccines, the approval of preservative-free multidose vials could further accelerate their availability and use.18 Ne w Technol o gies in Vaccine Produc tion The limitations of currently available vaccines, the complex manufacturing process, and the com- pressed production times underscore the need for more effective vaccines and more rapid, efficient, and reliable vaccine-production technologies, as well as considerably more surge capacity in the event of a pandemic. Multiple efforts are under way to address these areas, and new approaches to influenza-vaccine production as well as exist- ing technologies are summarized in Table 1. To be licensed, a new influenza vaccine must be shown to be safe and effective, to elicit anti- bodies, and to prevent influenza infection. Addi- tional studies may include correlating efficacy with less traditional immune responses (e.g., antibod- ies against NA or M2 or cellular responses) and comparing the efficacy of the new vaccine with that of a vaccine that has already been approved.19 The New England Journal of Medicine Downloaded from nejm.org by liang qiao on December 16, 2010. For personal use only. No other uses without permission. Copyright © 2010 Massachusetts Medical Society. All rights reserved. T h e n e w e ngl a nd j o u r na l o f m e dic i n e n engl j med 363;21 nejm.org november 18, 20102038 Interim Approaches Cell-Culture Techniques Our current egg-based vaccine-manufacturing pro- cess is vulnerable because of an insufficient egg supply in the event of a zoonotic outbreak of avi- an influenza or other diseases affecting chicken flocks and a lack of capacity for a surge in pro- duction. To better prepare us for seasonal influ- enza and the next pandemic, substantial resourc- es have been invested in developing mammalian cell cultures as an alternative substrate for the production of influenza vaccines with the goal of U.S. licensure in the near future.20 Although a shift from eggs to cell culture would have several advantages — allowing manufacturers to work directly with wild-type viruses, avoiding the gen- eration of egg-adaptive mutations in the HA pro- tein, increasing surge capacity in the event of a pandemic, and providing better manufacturing control through a closed-system fermentation pro- cess — limitations remain.21,22 For inactivated vaccines, large quantities of the viruses yielding sufficient HA protein would still need to be pro- duced. In addition, the cell-grown viruses need to be processed in a manner that is similar to the processing of viruses grown in eggs, so it remains to be seen whether cell-based technologies would substantially shorten the time needed to produce inactivated vaccines. In the United States, a cell- culture–based, live attenuated vaccine is also in late-stage preclinical development; however, since the viruses are not inactivated and are only mini- mally purified, studies to assess residual cell-sub- strate DNA are needed before they can progress to clinical testing.23 Adjuvants Adjuvants amplify the immune response to an an- tigen by enhancing the delivery and presentation of antigen as well as the recruitment of inflam- matory and immunocompetent cells to the area of antigen deposition, by directly activating an innate immune response, or both. Several HA- based seasonal influenza vaccines with adjuvants have been approved and used in Europe, includ- ing those formulated with phospholipids or oil- in-water emulsions.24,25 In 2009, H1N1 vaccines containing oil-in-water adjuvants were used in Eu- rope and other countries.26-28 Despite the approval and widespread use of such vaccines abroad and their excellent safety record, there were reserva- tions about adopting them in the United States. These concerns were expressed against a backdrop of caution toward vaccines in general among cer- tain segments of the public. Although it was for- tunate that adjuvants were not needed to enhance the immune response to the 2009 H1N1 vaccine, clinical trials have shown that oil-in-water adju- vants are needed to stimulate high levels of anti- bodies against influenza viruses that have novel HAs (e.g., H5N1 viruses), and these adjuvants may be critically important in future vaccination pro- grams.29-32 Purified bacterial outer-membrane pro- teins, toll-like receptors, and a variety of toll-like– receptor agonists (bacterial carbohydrates, lipids, proteins, and nucleic acids) have also shown prom- ise as next-generation adjuvants for influenza vac- cines, and several of these adjuvants are in the early stages of clinical testing.33-37 Novel Live Attenuated Vaccines Efforts also are under way to develop live influ- enza vaccines based on the influenza NS1 protein, a nonstructural, multifunctional protein involved Figure 1 (facing page). Structure and Replication Cycle of Influenza A Virus and Well-Characterized Adaptive Immune Responses. Eight gene segments code for 11 proteins, including hemagglutinin (HA) and neuraminidase (NA), which account for most of the known antigenic determinants. The portion of the matrix 2 (M2) protein outside the viral envelope is also antigenic. Adaptive immune re- sponses are shown in Panels A through D. In Panel A, the influenza HA protein mediates attachment of the virus to its host cell receptor. Antibodies directed against the HA protein block attachment of the virus to the host cell receptor or block fusion of the virus and host membrane. Antibodies generated against HA are correlated with vaccine protection against infection. In Panel B, antibodies generated against the NA protein do not prevent infection but limit the release of virus from infected cells. Antibodies directed against NA have been correlated with a reduction in disease sever- ity. In Panel C, antibodies generated against the highly conserved external domain of the M2 protein epitope interfere with virus assembly or constrain proton trans- port and are highly cross-reactive across virus sub- types. In Panel D, CD8+ T-cell responses to conserved influenza virus components have been correlated with enhanced clearance of virally infected cells; however, the exact degree to which they contribute to a reduc- tion in illness remains uncertain. NP denotes nucleo- protein, PA polymerase acidic protein, and PB2 poly- merase basic protein 2. Adapted from Kaiser 6 and Subbarao et al.7 The New England Journal of Medicine Downloaded from nejm.org by liang qiao on December 16, 2010. For personal use only. No other uses without permission. Copyright © 2010 Massachusetts Medical Society. All rights reserved. current concepts n engl j med 363;21 nejm.org november 18, 2010 2039 NA NA NA antibody M2 ion channel M2 ion channel Sialic acid receptors M1 matrix protein HA HA antibodies Nucleus Lipid bilayer Influenza virus Attachment to sialic acid receptors RNA synthesisEndocytosis and fusion M2 channel opens to permit proton entry, releasing genes A NA antibodiesB M2 antibodiesC NA antibodies bind to NA and prevent release of virus M2 antibodies can interfere with virus assembly or constrain proton transport Release No release Viral particles Epithelial cell H+ H+ H+ H+ Protein synthesis Packaging and budding Ribonucleo- protein assembly NA cleaves receptors, allowing virus release Cytokine productionCytolysis Activation T-cell receptor Peptide Influenza-specific CD8+ T cell Functional T cell Major histo- compatibility protein complex 1 PA PB2 NP Virus attachment to the host membrane through sialic acid receptors or fusion in endosomes is blocked Cell-mediated immunityD 1 Campion 10/28/10 AUTHOR PLEASE NOTE: Figure has been redrawn and type has been reset Please check carefully Author Fig # Title ME DE Artist Issue date COLOR FIGURE Draft 5 Lambert Knoper 11/18/10 Influenza structure and replication cycle The New England Journal of Medicine Downloaded from nejm.org by liang qiao on December 16, 2010. For personal use only. No other uses without permission. Copyright © 2010 Massachusetts Medical Society. All rights reserved. T h e n e w e ngl a nd j o u r na l o f m e dic i n e n engl j med 363;21 nejm.org november 18, 20102040 in viral replication and inhibition of the host’s innate immune responses. Preclinical studies have shown that infection with viruses containing an altered or deleted NS1 protein blocks viral repli- cation and stimulates both humoral and cellular immune responses.38,39 Early clinical data have shown that an intranasal NS1 vaccine is well tol- erated and gener ates neutralizing HA antibodies.40 Next Generation of Influenza Vaccines Although cell-based influenza vaccines and vac- cines containing adjuvants are likely to expand the capacity to produce influenza vaccines within sev- eral years, recombinant DNA techniques are fa- cilitating new production strategies by allowing vaccine candidates to be generated as soon as the genetic sequence of the influenza virus HA is known; this approach would eliminate the need to handle pathogenic viruses or to adapt viruses to grow in eggs or cells (Fig. 2). These technolo- gies, which are still mostly in early stages of development, may substantially reduce production timelines. Recombinant Proteins A recombinant trivalent HA protein–based influ- enza vaccine is in the late stages of clinical devel- opment in the United States (Fig. 2A). As soon as the influenza vaccine strains are selected, the genes encoding the HA proteins are cloned into bacu- lovirus vectors. Insect cells infected with these vectors express HA proteins, which are then fur- ther purified and formulated into a trivalent vaccine.41 The safety, immunogenicity, and effi- cacy profile of this vaccine has been reported, and an application has been submitted to the Food and Drug Administration for its approval Table 1. Current and New Approaches to Influenza-Vaccine Production. Vaccine Stage of Development Preclinical Development Phase 1 and 2 Clinical Testing Phase 3 Clinical Testing Licensed or Approved Inactivated vaccines Egg-based Yes Yes Yes Yes Cell-based Yes Yes Yes In Europe but not in the United States With adjuvant Yes Yes Yes In Europe but not in the United States Live attenuated vaccines Egg-based Yes Yes Yes Yes Cell-based Yes Yes No No Next generation Recombinant proteins Yes Yes Yes No Viruslike particles Yes Yes No No Viral vectors Yes Yes No No DNA-based vaccines Yes Yes No No Universal vaccines Yes Yes No No Figure 2 (facing page). New Approaches to Influenza- Vaccine Production. Steps in the production of vaccines using recombinant proteins (Panel A), viruslike particles (Panel B), viral vectors (Panel C), and DNA-based methods (Panel D) are shown. Once the sequence of an influenza virus genome is determined, recombinant DNA technology enables the rapid generation of influenza vaccines with the use of a variety of production strategies. In Panel A, the HA gene is cloned into a vector, which expresses the recombinant HA protein in infected cells, and the protein is subsequently purified. In Panel B, the simul- taneous infection of cells with individual vectors con- taining the HA, NA, and M1 genes, respectively, re- sults in self-assembling viruslike particles that contain HA and NA proteins on their surface but lack influenza gene segments. In Panel C, the influenza HA protein is expressed on the surface of a “carrier” virus that pre- sents it to the immune system but that cannot itself cause disease. In Panel D, a DNA vaccine consists of a DNA plasmid into which one or more influenza virus genes are inserted. HA denotes hemagglutinin, M1 matrix 1 protein, and NA neuraminidase. The New England Journal of Medicine Downloaded from nejm.org by liang qiao on December 16, 2010. For personal use only. No other uses without permission. Copyright © 2010 Massachusetts Medical Society. All rights reserved. current concepts n engl j med 363;21 nejm.org november 18, 2010 2041 HAHA NA M1 HAHA NA M1 HA HA NA A Recombinant proteins