Influenza: Status and Prospects for Its Prevention, Therapy, and Control

Pediatric Vaccinations: Update 1990

0031-3955/90 $0.00

+ .20

I nfl uenza: Status and Prospects for Its Prevention, Therapy, and Control Carole Heilman, PhD, * and John R. La Montagne, PhDt

Influenza, one of the ancient plagues of mankind, continues to produce epidemics in contemporary society. Accounts of epidemics of severe respiratory disease are found throughout history.36 These epidemics were often severe with extensive morbidity and mortality. The connection with cold weather and possibly other meteorologic phenomena is probably responsible for the name influenza, which it is thought comes from the Italian influentia or irifluentia di freddo. 20, 47 Unlike many of the other common infectious diseases which have tended to decrease in impact with the general improvement in sanitation and living standards, influenza epidemics continue to occur with predictable regularity and sometimes devastating if not catastrophic impact. Moreover, these epidemics affect all population groups and ages, apparently without discrimination. Influenza epidemics have been well described during the last century and at least five major pandemics have occurred since 1889 (Table 1).36 The often cited pandemic of 1918 and 1919 was clearly one of the worst epidemics of an infectious disease ever recorded. During this 2-year period it is estimated that the global toll was over 20 to 25 million deaths and perhaps billions of infections. It is difficult to realize the intensity of the epidemic that took more lives than World War I, which was just ending as this pandemic began. Many examples or anecdotes of the explosive nature of this epidemic have been written, but one example from Panati's description 47 is illustrative of the impact: Luxury ocean liners from Europe docked at New York harbor with up to seven percent fewer passengers than had embarked.

In the United States it is estimated that over 450,000 to 500,000 people died that fall and winter, with 187,000 deaths in the month of October From the Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland *Chief, Respiratory Diseases Branch tDirector

Pediatric Clinics of North America-Vol. 37, No.3, June 1990

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Table 1. Influenza Pandemics Since 1889 VIRUS TYPE

PANDEMIC PERIOD

1889-1891 1918-1919 1957-1958 1968-1969 1977-1978

H2N2 HINI H2N2 H3N2 HINI

? Asian-like Swine-like Asian or AlAichi/57 Hong Kong or AlHong Kong/68 Russian or AlUSSR/77

alone. 15, 19 These data provide poignant evidence of the power of influenza viruses to cause death and disease. In the interval since 1918, it is known that at least three pandemics have occurred. The first occurred in 1957 with the introduction of AlAs ian/ 57 (H2N2 or "Asian influenza"). * A second pandemic occurred in 1968 with the arrival of AlHong Kong/68 viruses (H3N2 or "Hong Kong influenza"). Approximately 10 years later AlUSSRl78 (HINI or "Russian influenza") viruses appeared. The appearance of HINI viruses in 1978 was totally unexpected and was important for two reasons. 50 First, it provided evidence that influenza virus types could recirculate in nature. This is important since HINI viruses had circulated earlier in the population, from 1918 to 1957 and were not isolated after the appearance of the H2N2 strains. In fact, the prototype Russian influenza virus isolated in 1977 was virtually identical to a virus that had circulated earlier, AlFort Monmouth/50 (HIN1), a virus responsible for epidemic influenza in 1950. 45 The second important aspect of the reappearance of HINI viruses is that since that time, HINI viruses have cocirculated in nature along with the H3N2 and influenza type B viruses. PROPERTIES OF INFLUENZA VIRUSES The viruses responsible for these massive epidemics are all members of the orthomyxovirust group of viruses. The orthomyxovirus group of viruses is distinguished by several important features, which are summarized in Table 2 and Figure 1 (for additional details see refs. 36 and 45). As indicated in the table, one important property of orthomyxoviruses is that their genome is segmented and of negative strand polarity.:j: The *The nomenclature of influenza viruses follows a simple convention based on the antigenic makeup of the two surface glycoproteins of the virus, the hemagglutinin (H or HA), and the neuraminidase (N or NA) glycoproteins. Three different types have been isolated from human cases during the last 50 years-HI or swine; H2 or Asian; and H3 or Hong Kong. Thus circulating viruses are designated as HINI or H3N2, etc. tThe term orthomyxoviruses is used to distingUish these viruses from a morphologically similar group of viruses called paramyxoviruses. Both virus types are of negative-strand polarity, are surrounded by a lipid membrane, and are pleomorphic in shape. However, the paramyxoviruses do not have a segmented genome and thus cannot undergo genetic reassortment, are larger in size and do not undergo antigenic shift or antigenic drift, and do not produce epidemics of the same magnitude as orthomyxoviruses. :j:Negative strand polarity indicates that the RNA molecule must be copied before it can be translated into a protein. Positive strand virus RNA can be directly translated. Poliovirus and rhinoviruses are examples of positive strand viruses.

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Table 2. Characteristics of Orthomyxoviruses Contain single-stranded RNA genes of negative strand polarity Pleomorphic virus particles approximately 80-124 nm in diameter Surrounded by lipid membrane that contains up to two viral glycoproteins-the hemagglutinin (HA or H) and the neuraminidase (NA or N) Genetic reassortment occurs readily Widely distributed in nature

segmented nature of the genome makes it possible for orthomyxoviruses to undergo genetic reassortment. Genetic reassortment is important in the evolution of influenza viruses because it is the mechanism responsible for antigenic shift.45 Antigenic shift and drift are two commonly recognized properties of the type A orthomyxoviruses. In contrast, only antigenic drift has been described for type B orthomyxoviruses (Table 3). Influenza C viruses are less well understood than either influenza A or B viruses. The C viruses appear to contain only one surface glycoprotein, which contains both the HA and NA-like or receptor destroying activities. 29, 36 The two surface glycoproteins of influenza A and B viruses are among the most heavily studied proteins in contemporary biology.36, 45 The threedimensional structure of the HA and NA of influenza A viruses has been solved and much is now known about the process of antigenic variation.lO, 57 The functions of these two proteins are extremely important. The HA is the protein on the virus that recognizes and binds to the viral receptor present on susceptible cells, in this way initiating the infectious process. A functional HA is required for the infectious process. Antibodies

Figure 1. An electron micrograph of influenza A virus particles that illustrates the pleomorphic nature of the particles. (x 500,(00) The spikes protruding from the membrane are the hemagglutinin and neuraminidase surface proteins. (From The National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland.)

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Table 3. Proteins of Influenza A Viruses

DESIGNATION

RNA SEGMENT LENGTH

MOLECULAR WEIGHT

PBl

2341

86,500

PB2

2341

84,000

PA HA

2233 1778

82,500 77,000

NP

1565

56,106

NA

1413

60,106

Ml

1027

27,861

M2

NSI NS2

15,000

890

25,825 14,216

FUNCTION

RNA polymerase protein, functions in early transcription, initiation RNA polymerase, host cell RNA cap binding activity, transcription RNA polymerase, chain elongation Hemagglutinin, binds virus to cell receptor, mediates viral/cellular fusion, virus entry into cell Nucleoprotein, binds to viral RNA, function unknown Neuraminidase, cleaves sialic acid residues from cell surface, facilitates virion particle release from infected cells Matrix or membrane protein, most common protein in virus particle, function unknown but thought to be structural Synthesized from a second open reading frame in Ml gene, function unknown, but mutants in this protein confer resistance to amantadine Nonstructural protein, function unknown Protein synthesized from second open reading frame found in the NSI gene, function unknown

to the HA will neutralize infectivity; however, the HA molecule possesses a plasticity that is quite remarkable. It can incorporate many amino acid changes without affecting its ability to recognize the cell receptor and also to mediate penetration of the cell through fusion of the viral and cellular membranes. 52 The HA molecule exists in the native state as a trimer, which is club shaped with its hydrophobic tail embedded in the viral membrane. The monomer consists of two proteins, called HAl and HA2, which are generated through proteolytic cleavage of a precursor HA molecule. This cleavage is required for infectivity and exposes a hydrophobic region buried within the stalk of the molecule which mediates the fusion between the viral and cellular membranes. Once this fusion step is completed the contents of the viral particle are emptied into the cell, and viral replication commences. The structure of the viral NA is also known. 10, 58 It is a tetramer. The NA is required to destroy the sialic acid residues present on the surface of cells and is thought to be particularly important in the exit of newly produced virions from the infected cell. The other viral proteins include the viral polymerase, the nucleoprotein (NP), the nonstructural protein (NS), and the matrix or membrane protein (Table 4). The viral polymerase is'made from the protein products of three of the viral genes (PB 1, PB2, and PA) and must perform two functions, the synthesis of complementary positive strand RNA (mRNA), which can be translated into viral proteins and the synthesis of the viral RNA (vRNA), which is subsequently incorporated into the virion. In addition, influenza employs a very interesting and unique mechanism for the synthesis of viral mRNA. The viral polymerase proteins (PBI and PB2) "capture" and utilize

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Table 4. Characteristics of Influenza A, B, and C Viruses

TYPE

NO. OF RNA SEGMENTS

NO. OF VIRAL PROTEINS

HA

NA

DRIFT

SHIFT

A

8

10

yes

yes

yes

yes

B C

8 7

10

7

yes yes

yes unk*

yes unk

no unk

D

?

?

?

?

?

*

=

?

HOST RANGE

Humans, horses, pigs, seals, whales, migrating waterfowl Humans Humans Tick-borne

unknown

the methylated and capped 3' ends of nascent cellular mRNAs and add these captured fragments to viral mRNAs so th~t they can then be translated by the cellular ribosome. 39 This occurs early in infection. To accomplish these tasks the influenza virus particle contains functional viral RNA polymerase. The nucleoprotein or NP is associated with the viral RNA molecule, but its function is not known. Two NS proteins are also produced. The precise functions of these two proteins have not been defined, but they are related to viral RNA synthesis. Finally, two M proteins are synthesized by the virus. Ml is the most common protein of the virion particle and is thought to be an important structural protein. M2 is synthesized from the same gene but from an mRNA synthesized that contains a small part from the 3' end of the Ml mRNA and a larger section from a second open reading frame on the M gene that is copied. These two different sections are spliced together to form the M2 RNA. The M2 protein is believed to be important in the assembly and morphogenesis of the virion particle.

EPIDEMIOLOGY OF INFLUENZA VIRUSES It is known that at least three major antigenic types of influenza virus circulate in humans. A fourth viral type, designated as influenza D, has also been described. 11 Little is known about the D type viruses other than they are vector-borne (ticks) and have not been clearly associated with illness in humans. As mentioned earlier, the only known host for both the influenza Band C viruses is the human. In contrast, influenza A viruses are widely distributed in nature (Table 5). The illness produced by influenza A and B is difficult, if not impossible, to distinguish clinically. In contrast, the illness associated with influenza C infections is generally milder and much less frequently observed. All three types are transmitted via aerosol and produce severe respiratory disease with sudden onset that usually is accompanied by prostration, fever, and other constitutional signs and symptoms. Why then is influenza A the virus most commonly associated with epidemics and pandemics? One explanation for this difference is the capacity of influenza A viruses to infect and cause disease in a growing list

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Table 5. Distribution of Influenza Viruses in Nature HEMAGGLUTININ ITPES

Humans Pigs Seals Seagulls Chickens Turkeys Ducks

HI, H2, H3 HI, H2 H4, H7 Hl3 H3, H4, HS, H6, H7, HI, H4, HS, H6, H7, HI, H2, H3, H4, HS, H8, H9, HlO, Hll,

H9, HlO H9, HlO H6, H7, HI2

NEURAMINIDASE ITPES

NI, N2 NI, N2 NS, N7 NlO NI, N2, N3, N4, N7, N8 NI, N2, N3, N4, N7, N8 NI, N2, N4, NS, N6, N7, N8, N9

of animal species, including some, such as pigs, horses and migratory birds, with frequent contact with humans. The role that these animals play in the epidemiology of influenza is important in that they not only can amplifY the viruses in circulation in the human population, but they also can serve as a reservoir for new strains and a point in which reassortment can occur. This wide distribution and the facility with which genetic reassortment can occur permit genetic exchange between different antigenic types. Genetic reassortment is restricted in that it has been observed only between different influenza A or influenza B viruses, but never between A and B or C viruses. It is now thought that this restriction is due to several factors, which include the fact that the genomes of the three virus types are organized differently and that the RNA segments are of different sizes. Consequently, a genetic barrier exists to successful reassortment. The property of genetic reassortment is also extremely important in vaccine production, research, and development, since genetic reassortment is used to provide useful properties to viruses, such as attenuation, or to produce high-yield strains for production of the inactivated vaccine. The experience with influenza epidemics reveals that not all segments of the population are affected equally.22 High-risk groups for influenza include persons with chronic lung (including asthma) or cardiac disease, diabetes, and otherwise healthy persons age 65 years or older. It is less well appreciated that influenza produces serious problems in pregnant women, particularly during the last trimester, and in infants. For example, the 1957 pandemic of Asian flu was associated with significant excess mortality and morbidity for pregnant women. 36 In addition, it has been reported that influenza infections are responsible for 25% of the hospitalizations of infants during the winter months. 23 Because of this toll of infection and disease, these two groups should be actively considered for immunization. ANTIGENIC SHIFT AND DRIFT Two forms of antigenic variation have been observed among influenza viruses. One form, called antigenic shift, has been alluded to earlier. In this case, influenza viruses acquire a new antigenic form of one or both of the surface glycoproteins-the HA and/or NA. This change is thought to occur through genetic reassortment. The emergence of the H3N2 family of influenza A viruses is thought to have occurred through the genetic

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reassortment of the then circulating Asian virus (H2N2) and an equine or avian virus containing the H3 HA.4O There is also evidence that influenza viruses recycle so that previously prevalent antigenic forms "disappear" only to emerge many years later. 50 This has been suggested as the source of the H1N1 influenza virus (Russian flu) that reappeared or recycled in 1977. The molecular and antigenic similarities of the 1977 virus with earlier H1N1 viruses demonstrated that recycling of antigenic types was possible. The second form of antigenic variation is called antigenic drift. It is a much more common manifestation of antigenic variation among the influenza A and B viruses. This process occurs through the constant accumulation of single-point mutations in the HA and NA genes that are selected by the immunologic background in the population. These mutants permit influenza A and B viruses to evade immunologic defense mechanisms effectively and to propagate in individuals who have preexisting immunity to other strains, even closely related strains. Antigenic variants are constantly being produced, but typically one or two predominate during any given epidemic. It is this capacity for antigenic variation and change that makes the development of vaccines to prevent influenza infections a particularly difficult challenge. The current practice is to incorporate important new antigenic variants into the influenza vaccine each year. This is the only vaccine in widespread use the antigenic composition of which is changed continuously to accommodate these new variants.

CLINICAL MANIFESTATIONS OF INFECTION Influenza viruses produce a varied clinical response in humans. 36, 45 The clinical manifestations of infection are moderated by several factors, including the general health of the individual, the virulence or pathogenicity of the virus, and the previous experience of the individual with influenza (i.e., the immune status). However, the usual presentation of influenza in adults is one of a rapid, sudden onset of fever, vague nasal symptoms that can include coryza, an unproductive cough, myalgia, and headache. The illness is rapidly prostrating and usually lasts from 3 to 5 days. Recovery is usually complete and uneventful. In infants and children the illness differs in that rhinitis and gastrointestinal symptoms are more commonly reported. In addition, infants are likely to have fever for a longer period and to shed virus for longer periods. Complications of influenza include pneumonia, either a primary viral pneumonia or a secondary bacterial pneumonia. This is a very serious complication of the infection and is probably responsible for most of the deaths associated with influenza. Direct infection by the virus of other organ systems is rare, since the virus rarely produces a viremia. The infection is particularly severe in asthmatics and other individuals with compromised pulmonary or cardiovascular function. Influenza B has been rarely associated with rhabdomyolysis and severe myopathy and nephropathy. Acute transient crural myopathy, acute myopathy with rhabdomyolysis, and myoglobinuria with renal dysfunction and primary viral pneumonia have been described in children. 36 Reye's syndrome, a rare

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complication of influenza (primarily influenza B) infection in children up to 18 years of age, has been associated with the use of aspirin or other salicylate-containing medications to control fever during the acute phase of the infection. 3o The pathogenesis of Reye's syndrome is unknown. Pregnancy also has been found to be an underlying risk factor for severe illness and death, particularly during pandemics. The risk in pregnancy is most closely associated with women in their third trimester and is probably associated with the hemodynamic changes that occur late in pregnancy. 36 Diagnosis of influenza is most easily made in the context of an epidemic. Direct viral isolation from nasopharyngeal washings into tissue culture, either Madin-Darby Canine Kidney (MDCK) or primary rhesus monkey kidney, primary chick kidney, or directly into the allantoic cavity of chick embryos, is the usual practice. Direct examination of nasopharyngeal material using immunofluorescent techniques or other forms of immunoassays also is used, and several such tests have been described. One problem with these techniques is that the results are not immediately available and are therefore not useful guides to therapy, which must be initiated promptly for maximal benefit. Direct measurements of acute and convalescent sera for antibodies is of epidemiologic value, but of little value in the acute clinical management of the infection. IMMUNOLOGIC RESPONSE TO INFLUENZA ANTIGENS Many studies have documented that influenza infection stimulates strong type-specific immunity. This immunity is focused primarily on the surface glycoproteins and particularly against the HA of influenza viruses. The recent reintroduction of the HINI virus in 1977 to 1978 illustrates this point very well. As mentioned earlier, the HINI virus that first circulated was very similar to viruses that circulated before 1957. Epidemiologic studies demonstrated that influenzal illness did not occur in individuals born before 1957, although exposure was commonY Symptomatic illness due to HINI viruses is still rare in the older cohort, whereas it is common in young adults and children. Immunologic responses to other viral antigens are also important. Immunity to the NA may have a dampening effect on infection resulting in a moderated infection. In addition, immunity to other viral proteins, such as the NP protein, has been related to antibodydependent cell-mediated cytotoxicity (ADCC). However, immunity to influenza has been most closely related to the presence of mucosal antibodies, such as IgA present in the mucosal surfaces where infection occurs. 45 Importantly, the immune response to influenza antigens is modified by the person's experience, either through vaccination or natural infection, with other influenza antigens earlier in life. This aspect of the immune response to influenza antigens is called "original antigenic sin. "21 As a consequence, individuals will respond with a heightened immune response to crossreactive determinants from earlier influenza virus exposures. This effect may contribute in some way to the lack of complete protection observed with influenza vaccines. However, many studies have confirmed that the inactivated vaccine does provide significant protection to the individual, although it is clear that even though immunization is common, epidemics

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still occur. In the high-risk elderly group, influenza vaccines appear to reduce mortality significantly but do not appreciably reduce morbidity from influenza. 25

APPROACHES TO VACCINATION AGAINST INFLUENZA

II 'i"

Two basic approaches for immunization have been pursued: the use of inactivated virus preparations and the use of live, attenuated viruses. Both avenues of research have been pursued since the influenza viruses were first isolated in 1933 by Smith et al. 53 At the present time only vaccines prepared with inactivated or killed virus particles are licensed for use in the United States. Although these vaccines have evolved enormously since they were first used experimentally during World War II, they still are essentially produced by growing the virus in the allantoic sac of embryonated chick eggs, purifying the virus from the allantoic fluid, and inactivating the virus with a chemical agent, usually formalin. Numerous refinements have been introduced into this process over the years, and present-day inactivated or killed vaccines are quite different from their predecessors. Four major innovations have been incorporated: the use of zonal centrifugation, the use of ether or other lipid solvents to disrupt the virus, the introduction of high-yield reassortants to improve yields in the chick embryo, and the development of better methods to quantitate the amount of viral antigens present in the vaccines. All of these efforts have led to inactivated vaccines that are better purified and more predictable in their reactogenicity and immunogenicity.61 The process of influenza vaccine production is still a difficult one because the formula usually is changed each year. The economic incentives are high to improve the yield of viral particles by the use of either improved high-yield reassortants or by the application of techniques that eliminate the use of the embryonated chick eggs as the substrate for influenza virus production. The inactivated influenza vaccines have been repeatedly shown to be approximately 70% effective in preventing influenza. 31 The efficacy is directly related to the fidelity with which the vaccine strain matches the epidemic strain. This is a difficult task since the vaccine strains must be selected the year before the epidemic appears. In addition, recent reports51 suggest that passage of human strains through the chick alters the antigenic properties of the HA. It is argued that this may reduce the efficacy of the vaccine. Variations on this use of inactivated antigens include the use of vaccines containing only NA,37 the so-called infection permissive vaccines. The use of a NA vaccine, it is argued, would permit limited replication by wild strains, which would stimulate a more substantive and long-lasting humoral and local immunity to influenza. Early attempts to develop NA vaccines relied on the use of reassortant viruses. Early trials with NAspecific vaccines did provide evidence that these vaccines stimulated immunity that permitted infection. Subsequent efforts to develop NA vaccines have relied on the use of recombinant DNA methods to provide purified NA proteins. The current vaccine formulation in use in the United States contains

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equal amounts (15 f.1g) of each of the three circulating virus types (HINl, H3N2, and B). It is recommended that individuals with conditions that place them at high· risk for influenza (Table 6), healthy persons 65 years of age or older, and persons with certain chronic disease receive annual immunization with inactivated influenza vaccine. In addition, people capable of transmitting the infection to high-risk individuals should also be vaccinated. This includes physicians, nurses, and other health care workers. The vaccine is updated annually to reflect changes in the antigenicity of circulating viral strains in an effort to make it more representative of contemporaneous viral strains. These viruses are identified by an international network of laboratories that maintain surveillance for new influenza variants throughout the world. These laboratories are coordinated through the World Health Organization.

ATTENUATED INFLUENZA VACCINES The development of an attenuated influenza vaccine has been a long sought after objective. In some countries, particularly Europe until 1978 and the Soviet Union, attenuated influenza vaccines have reached wide usage only to fall back into disfavor. The major difficulty has been in generating attenuated vaccines that are reproducibly attenuated and safe. The approaches pursued toward the generation of attenuated vaccines are shown in Table 7. One of the most promising research efforts has been development of the live attenuated, cold-adapted (ca) influenza virus reassortant vaccine. This vaccine relies on the use of an attenuated donor virus to confer the property of attenuation to contemporaneous wild type strains by genetic Table 6. Recommendations for Influenza Vaccination Persons who should receive influenza vaccine include the following: • Healthy individuals 65 years of age or older; • Adults or children with underlying chronic pulmonary or cardiac diseases, especially asthma, chronic obstructive pulmonary disease, bronchopulmonary dysplasia, and cancer; • Residents of nursing homes and other chronic care facilities; • Persons with chronic metabolic diseases, including diabetes mellitus, renal dysfunction. hemoglobinopathies, and immunosuppression, and; • Children or teenagers receiving chronic aspirin therapy. • Persons capable of transmitting influenza to individuals at high-risk for influenza, including: physicians, nurses, health care workers, providers of home care for high-risk persons, and family members of high-risk individuals. Vaccine Administration • Vaccine should be given in the fall, before the influenza season starts. • Vaccine should be given to adults and older children in the deltoid muscle and in infants and young children it should be given in the anterolateral aspect of the thigh musculature. Vaccine dosage recommendations Age Dose Number of doses Two doses, administered 4 or more weeks apart 6-35 mo 0.25 ml (7.5 fLg) Two doses, administered 4 or more weeks apart 3-12 yr 0.5 ml (15 fLg) >12 yr 0.5 ml (15 fLg) One dose

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Table 7. Approaches to the Development of Attenuated Influenza Vaccines APPROACH

Host-range mutants

Cold-adapted strains

Avian-human Temperature sensitive

STATUS

The use of host-range mutants has been attempted several times but has now been abandoned because it was impossible to predict attenuation. These vaccines are generated through genetic reassortment with a attenuated donor virus, AlAnn Arbor/6/60 (H2N2). The ca vaccines have been shown to be reproducibly attenuated and safe. Efficacy trials are in progress. Reassortants using avirulent avian donors have not been shown to be reproducibly attenuated. Temperature sensitive (ts) mutants of H3N2 viruses were found to be attenuated and were used to generate reassortants with contemporaneous viruses. These reassortants were shown not to be predictably attenuated.

reassortment. It has been shown that reassortants that contain the six internal virus genes that confer the property of growth at 25°C (Le., cold adaptation) and the two genes from the currently circulating influenza virus strain, which code for the external influenza proteins, the HA and NA, are genetically stable. A series of such reassortants have now been evaluated in over 20,000 people between the ages of 4 months and 80+ years of age and found to be safe, genetically stable, and immunogenic. This live, attenuated vaccine is very stable, owing partly to the multigenic requirement for the attenuated phenotype. 54 Because the ca virus is limited in its replication at temperatures below 37°C, the virus vaccine, which is administered intranasally, can only replicate at the site of administration. Local antibody response to virus replication, specifically IgA antibody production, is the major mechanism by which this vaccine affords protection against influenza. The safety of the ca A vaccine (Le., containing external genes from either H3N2 or H1N1 influenza A viruses) has been repeatedly demonstrated when given at doses that will achieve approximately 100 human infectious doses (HID). In adults, some degree of reactogenicity, as measured by excess frequency of mild respiratory symptoms, is routinely seen in 8 to 10% of the adult ca vaccine recipients. At doses above the recommended levels, fever and myalgia are observed in some vaccine recipients. 6 In children receiving the recommended dosage of vaccine, no discernible clinical illness associated with the ca A vaccine has been observed. rhe long- and short-term safety of the ca A vaccine in volunteers at high risk to complications with influenza virus infections (L e., the elderly, asthmatics, cystic fibrotics) has also been demonstrated (Atmar R: Personal communication, 1989).24.35,37 Clinical studies in adults, children, and infants with ca B vaccines (Le., containing external genes from influenza B viruses), although limited, have confirmed their overall safety and lack of reactogenicity (Edwards K: Personal communication; Belshe R: Personal communication). Limited replication, approximately 100-fold less than wild-type virus, of both ca A and ca B vaccine virus is observed following vaccine

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administration to seronegative individuals. In children, low levels of virus shedding may continue for up to 10 days after vaccine administration although no evidence for transmission or reversion of the ca virus has ever been detected. 4, 1.3, 16,44,63,65 The immunogenicity of the ca A vaccine has been extensively evaluated. When the antibody responses in adult volunteers receiving either inactivated or ca vaccines are compared, both are found to elicit similar antibody responses, although the predominant class of responding antibody differs. In general, higher levels of serum IgG antibody responses are observed in recipients of inactivated vaccine, whereas ca vaccine recipients mount higher levels of nasal IgA antibodies. The duration of the antibody response in adult vaccinees receiving the inactivated or the ca reassortant vaccines is approximately 6 months. 16 Antibody profiles following ca or inactivated vaccine administration in children not previously exposed to influenza virus are quite different from those seen in the adult population. Unlike ca vaccination in adults, significant serum antibody responses are seen in the majority (60-85%) of children receiving ca vaccine. Serum IgA and local IgA antibodies are seen almost exclusively in children receiving the ca vaccine, while serum IgM antibodies are more frequently associated with recipients of the inactivated vaccines. The duration of immunity between seronegative children receiving either the ca or the inactivated vaccine is significantly different. In general, the serum antibody response, measured by hemagglutination inhibition (HAl), is detected 4 to 6 weeks after ca vaccination and remains at approximately the same level over a period of 1 to 2 years. Children previously not exposed to influenza virus who were vaccinated with inactivated vaccine show a duration of immunity similar to that seen in adults, approximately 6 months. 62 The correlates of protection for recipients of ca A and inactivated influenza vaccines has been comparatively evaluated. In the adult population receiving inactivated vaccine, protection against influenza infection or illness correlated with the level of serum NA-inhibiting antibody, serum HAl antibody, and nasal IgG antibody to the viral HA. The protective correlates in the adults receiving ca A vaccine were markedly different however. In this population, protection against virus infection and illness correlated with nasal IgA antibodies to the HA and serum NA-inhibiting antibodies. In ca vaccinees, no correlation was observed between protection and the level of HAl serum antibodies. This observation suggests that alternative immune mechanisms are oapable of conferring resistance to influenza infection and illness. 9 The protective role of nasal IgA has also been demonstrated in children. 32 To assess the relative protection of the ca and the inactivated vaccine several large field studies have been and currently are being conducted. Between 1978 and 1980 the protective efficacy of two monovalent ca A vaccines was evaluated in college students. Although the overall frequency of respiratory infections was the same when the vaccine and the placebo groups were compared, influenza-specific infections and illnesses were reduced significantly in the vaccine group suggesting vaccine-induced protective efficacy during an influenza outbreak. When the overall efficacy of the ca A and inactivated vaccine groups were compared, the data

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suggested that both vaccines were equivalent. 13 Preliminary studies in a small number of children receiving either inactivated or ca vaccine and then followed during two influenza seasons have suggested a greater efficacy of the ca vaccine in the young age group (Couch R: Personal communication, 1989). Additional studies are in progress. Although it appears that the ca vaccine is at least as good as the inactivated vaccine in a population previously exposed to influenza virus, the real value of the ca vaccine may be in the administration of this vaccine to young children. The data consistently demonstrate that the ca vaccine is extremely safe and efficacious in this age group. Children, based on epidemiologic data, appear to be a major factor in the spread of influenza disease. In addition, they suffer unnecessarily high degrees of morbidity during yearly epidemics. Experiences with the mass vaccination of children in Japan and the Soviet Union have suggested a value in this approach for the control of epidemics. 46• 48 Thus, the ca vaccine approach, in children, appears to offer several advantages not found with the administration of inactivated vaccines including ease of administration, no demonstrated reactogenicity, long-lived immunogenicity, and greater protection.

CHEMOPROPHYLAXIS AND CHEMOTHERAPY OF INFLUENZA INFECTIONS The availability of effective antiviral compounds for the prevention and therapy of influenza infections has been a relatively recent development. Two related adamantanamines, amantadine and its derivative rimantadine, have been shown to be effective in the prophylaxis and treatment of influenza A infections. Only one, amantadine is currently licensed for use in the United States. Other compounds, such as ribavirin, have not been evaluated completely in humans and the effectiveness of these substances needs to be established more firmly. The adamantanamines appear to exert their antiviral effect on the same viral target since viral mutants resistant to amantadine are also resistant to rimantadine. Both of these compounds appear to inhibit late steps in the replication of influenza A viruses, particularly assembly of the viral membrane. This effect is thought to occur by inhibiting a virus-specific process, mediated by viral protein M2, that maintains the acidity of the microenvironment of the Golgi above pH 5.5. In the presence of either drug the pH goes down and, it is postulated, an irreversible conformational change occurs in the HA which makes the virus noninfectious. 3 Clinical trials have demonstrated that both compounds exert a significant chemoprophylactic effect. 17, 18 In one study, conducted by Dolin et al. 17 rimantadine and amantadine had approximately the same effect in preventing influenza A (HINl) infections in a college student population. Differences Were noted, however, in the frequency of side effects between these two compounds and the placebo control group. Rimantadine was found to be essentially no different than placebo while a significant fraction of the amantadine-treated subjects withdrew from the study due to an inability to maintain amantadine therapy, It is this feature that distinguishes

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amantadine from rimantadine most powerfully. The toxic effects of amantadine are usually any of a variety of central nervous system (eNS) manifestations, including vertigo, difficulty concentrating, sleeplessness, and excitability. These reactions are dose dependent and are directly related to blood levels of amantadine in circulation. They disappear when therapy is stopped. Because amantadine is not metabolized and is excreted entirely by the kidneys, precautions must be taken in using this drug in people with compromised kidney function. Table 8 describes the current recommendations for amantadine use in the United States. Rimantadine, on the other hand, is metabolized and does not produce the associated side effects seen with amantadine. In addition, it appears to have pharmacologic properties that may enhance its activity, particularly the concentration of the drug in mucous secretions. Finally, rimantadine has a plasma elimination half-life that is two times longer than that for amantadine. 18 Resistance to these drugs is easily obtained in the laboratory. Resistant mutants have been carefully studied and it is now clear that point mutations to the amino acids in positions 28 to 31 of the M2 protein of influenza are associated with resistance. 27 The occurrence of resistant viruses from humans 26 and from animals 2 treated with these drugs also has been reported. The clinical significance of these resistant mutants in humans is unknown. Recent work by Hayden 28 suggests that resistant variants can be transmitted within families in which these drugs were being used in a postexposure setting. Other studies indicate that treatment of families and contacts clearly provides prophylactic benefit59 and provided no evidence that these viruses were transmitted to other family members. These factors suggest that routine use of rimantadine or amantadine for postexposure prophylaxis in families should be carefully considered. The effectiveness of both drugs given as therapeutic agents has also been studied. Both compounds produce an antipyretic effect in influenza A infections, presumably due to the direct antiviral effect of the drugs. 12 It is, however, difficult to demonstrate a clear beneficial effect of the drugs because of the self-limiting nature of influenza infections. Despite this problem, several studies 18 have shown a reduction in the severity of infection Table 8. Recommendations for the Use of Amantadine (Symmetrel) AGE GROUP

No recognized renal disease 1-9 years lO-64 years ~64 years Recognized renal disease Creatinine clearance (mUmin-1.73 mL) ~80

60-79 40-59 30-39 20-29 10-19

DOSAGE

4.4-8.8 mg/kg per day once daily or divided twice daily, total dosage should not exceed ISO mg/day 200 mg once daily or divided twice daily lOO mg once daily

100 200 100 200 100 200

mg twice daily mg/l00 mg on alternate days mg once daily mg twice weekly mg thrice weekly mg/l00 mg alternating every 7 days

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as assessed by the comparison of clinical scores. In addition, individuals treated with these compounds returned to work or school sooner than the untreated groups. The relative effectiveness of these two drugs in preventing or treating the complications of influenza, particularly viral pneumonia, is not known. Amantadine, and rimantadine if it becomes available, are best used as adjuncts to the use of vaccines to prevent influenza A infections and in therapy of acute infections. Current recommendations for amantadine call for its use in outbreak situations in nursing homes, for example, in such a way as to protect vaccine recipients during the period immediately after immunization before they have developed protective levels of antibodies. Clearly, one of the factors that greatly complicates the use of these antivirals is the lack of rapid viral diagnostic methods that physicians could quickly use to diagnose the infection. Until these become available, it will complicate the use of these drugs therapeutically, except during epidemics of influenza. PROSPECTS FOR FUTURE ADVANCES IN THE DEVELOPMENT OF VACCINES AND THERAPEUTIC AGENTS TO PREVENT, TREAT, AND CONTROL INFLUENZA The evaluation of current, as well as new, methods for the production and delivery of influenza virus vaccines remains an area of intensive research. Several areas that deserve selective mention include the evaluation of our current system of producing influenza virus vaccines in eggs, attempts to enhance the immunogenicity of inactivated vaccines through the use of adjuvants or alternate routes of immunization, and potential avenues for the development of recombinant influenza virus vaccines. Influenza viruses used in the production of inactivated vaccines are grown in eggs. Mounting evidence that the host cell substrate in which the virus is grown can exert a selective mutational pressure on the original virus isolate recently has resulted in a careful evaluation of the effects of host cell substrate on the antigenic properties of influenza viruses. Using a single throat wash sample from an influenza A/H3N2 infected patient, Katz and Webster demonstrated the presence of at least four classes of influenza subtypes when a portion of this sample was grown in embryonated chicken eggs, but only a single virus subtype was obtained if a portion of this same sample was grown in mammalian cells. 33 The relationship between the eggderived variants and the mammalian-cell derived variant was further evaluated. Ferrets were immunized with either 7 JLg viral HA or 21 JLg viral HA of formalin-inactivated influenza virus grown in either eggs or mammalian cells. The ferrets were then challenged with "homologous" wild-type virus propagated in either eggs or mammalian cells. Vaccination with either the low- or high-dose mammalian cell-derived formalin-inactivated vaccine provided similar protection when ferrets were challenged with either the egg-grown or the mammalian-cell-grown wild type virus (75-100%). In contrast, all ferrets that were vaccinated with the lower dose of egg-derived formalin-inactivated vaccine were not protected against

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infection when subsequently challenged by mammalian-ceIl-derived wildtype influenza, even though protection (63%) was observed when challenged with egg-grown wild type virus. At higher doses of egg-grown vaccine, protection of the egg-grown vaccinees against challenge with either the egg-derived or the mammalian-ceIl-derived was observed, although the level of protection was less (44-50%) than that observed with the animals vaccinated with the mammalian-ceIl-derived vaccine. 34 These results suggest that virus grown in mammalian cells may afford a broader range of protection in addition to being better immunogens when compared to vaccines made from eggs. Further research in this area is needed to better assess the impact of this phenomenon on influenza vaccines used in human populations. The use of adjuvants to enhance the immunogenicity of inactivated influenza virus vaccine is currently under increased developmental activity by several vaccine manufacturers. As such, much of the research in this area has remained proprietary. Several reports suggest, however, the value of continuing basic research in this area. Perhaps one of the most promising classes of adjuvants currently under study is N-acetylmuramyl-L-alanyl-Disoglutamine (MOP) and its derivatives. MOP has been widely used as an alternative component for mycobacterial cells in Freund's adjuvant. Thus, the safety and immunomodulating properties are well known. In the mouse and guinea pig models, subcutaneous administration of MOP derivatives, in combination with inactivated influenza vaccine, was shown to increase levels of influenza antibodies when compared to vaccine alone. 57 The lack of reactogenicity is encouraging. Other adjuvants are also under consideration. Profeta et al. 49 described a clinical trial to evaluate the immunomodulating activity of RU41740, a glycoprotein from Klebsiella pneumoniae, when used in conjunction with influenza vaccination. When evaluated in a group of volunteers over the age of 65, having prevaccination HAl antibody titers of less than 20, those volunteers who received by mouth 4 mg of RU41740 daily for 14 days postvaccination demonstrated a significant increase in GMT titers to both influenza A vaccine strains when compared to those who did not receive the adjuvant. Tamura et al. 55,56 have evaluated the cholera toxin B (CTB) subunit's ability to enhance protection against influenza virus infection in Balb/C mice. Using an intranasal delivery system to administer inactivated vaccine, high levels of both nasal IgA and serum HAl antibodies were observed, in a dose-dependent manner, in animals inoculated with both the inactivated influenza virus vaccine and CTB. This increased antibody response correlated with protective efficacy. In addition, antibodies produced provided protection against subsequent challenge with either influenza A HINI and influenza A H3N2 subtypes. The oral route of inactivated vaccine administration has also been reevaluated with the emphasis on the ability to stimulate intestinal IgA cells, which in turn would migrate via the bloodstream to the respiratory tract. In the mouse, oral administration of inactivated influenza vaccine or live virus with or without CTB stimulates IgA and IgG specific responses in the respiratory tract, without a concomitant rise in serum antibody levels. These antibodies were protective when mice were challenged with wild type virus. 5 , 7 The

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level and duration of antibody response could be further increased when the orally administered inactivated antigen was microencapsulated. 43 Alternative vaccine approaches using recombinant DNA methodology also are under evaluation. The use of baculovirus expression vector has theoretic advantages in that insertion of a foreign gene into the polyhedron gene site results in the expression and translation of large amounts of the inserted gene. Possee48 successfully expressed the hemagglutinin of influenza virus in insect cells using this vector system. Although optimal expression was not achieved, the ability to express and transport a glycoprotein using this system suggests that further studies may be appropriate. Although the use of recombinant vectors containing defined regions of influenza dsDNA is commonplace, the research emphasis has focused on using this approach as a tool to further understanding the function(s) of defined regions of the influenza virus genome. In this respect, recombinant approaches have been successful in identifYing the precise regions of the virus proteins responsible for mounting a T cell response to infection. These data should prove invaluable in the development of alternative vaccines. The use of recombinant approaches to evaluate the ability of antisense RNA (RNA that is the complementary to mRNA) to inhibit influenza virus replication, although technically successful, has demonstrated that this approach for vaccination is not a viable alternative. Briefly, the selective expression of antisense RNA has been used to actively inhibit the gene expression. Using recombinants to express selectively the antisense RNA of influenza virus, these recombinants were shown to have no influence on the replication of influenza virus and thus may be of no value as an alternative vaccine approach. 41 Of most recent interest has been the potential use of a modified influenza virus as an expression vector.42 Two future possibilities of this approach are immediately apparent. First, this technology theoretically allows for a simple and controlled modification of an influenza virus such that improved inactivated vaccines may be constructed in addition to new live, attenuated vaccines. Second, this technology implies that a live, attenuated influenza virus may not only be constructed for use as a vaccine but may also be useful as a vector for other respiratory antigens. For example, the insertion of the sequences for the glycoprotein or fusion proteins of the respiratory syncytial virus (RSV) into this modified influenza virus may allow for the antibody response to both the influenza virus and the RSV to be achieved. Finally, the use of molecular biology as a tool to understand the retrospective and prospective relationship among influenza viruses will become increasingly more important in forecasting epidemic variants. The history of influenza indicates that the unexpected is the norm. It will take a continued commitment to research on influenza to provide the tools needed to confront this problem and, it is hoped, improve our ability to prevent and treat this ancient plague. CONCLUSION Influenza is a major public health problem. Infections due to influenza viruses are responsible for major epidemics of respiratory disease each year.

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In this article the important biological, molecular, and epidemiologic characteristics of influenza A, B, and C viruses are discussed. Approaches to the prevention and control of these important viruses through vaccination or therapy are briefly described. In addition, the important new areas of vaccine research are summarized, with an emphasis on the status of the development of live, attenuated influenza vaccines and the current issues related to the use of amantadine for the therapy or chemoprophylaxis of influenza.

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Address reprint requests to Carole Heilman, PhD National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD 20892