2009 대유행 인플루엔자 바이러스에 대한 임상적 측면에서 세계보건기구 자문단의 집필위원회(the Writing Committee of the World Health Organization (WHO) Consultation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza)가 [뉴잉글랜드저널오브메디신]에 기고한 “2009 신종플루 바이러스 감염의 임상적 측면”이라는 리뷰 아티클입니다.

WHO에 대한 비판적 견해가 거의 반영되지 않은 글이고, 타미플루 독점판매권을 가지고 있는 로슈 및 릴렌자 판매사인 글락소스미스클라인, 그리고 백신 제조사인 백스터, 노바티스, 글락소스미스클라인 등으로부터 연구비, 강연료, 자문료 등의 명목으로 재정적 지원을 받은 바 있는 일부 전문가들이 집필위원회에 포함되어 있다는 점도 고려해서 리뷰 아티클을 읽어야 할 것 같습니다.

Volume 362:1708-1719 May 6, 2010 Number 18 Next 출처 : http://content.nejm.org/cgi/content/full/362/18/1708

During the spring of 2009, a novel influenza A (H1N1) virus of swine origin caused human infection and acute respiratory illness in Mexico.^1,2 After initially spreading among persons in the United States and Canada,^3,4 the virus spread globally, resulting in the first influenza pandemic since 1968 with circulation outside the usual influenza season in the Northern Hemisphere (see the Supplementary Appendix, available with the full text of this article at NEJM.org). As of March 2010, almost all countries had reported cases, and more than 17,700 deaths among laboratory-confirmed cases had been reported to the World Health Organization (WHO).⁵ The number of laboratory-confirmed cases significantly underestimates the pandemic’s impact. In the United States, an estimated 59 million illnesses, 265,000 hospitalizations, and 12,000 deaths had been caused by the 2009 H1N1 virus as of mid-February 2010.⁶ This article reviews virologic, epidemiologic, and clinical data on 2009 H1N1 virus infections and summarizes key issues for clinicians worldwide.

Viral Characteristics

Pandemic 2009 H1N1 virus derives six genes from triple-reassortant North American swine virus lineages and two genes (encoding neuraminidase and matrix proteins) from Eurasian swine virus lineages.⁴ Although the 2009 H1N1 virus is antigenically distinct from other human and swine influenza A (H1N1) viruses,⁴ strains of this virus have been antigenically homogeneous, and the A/California/7/2009 strain that was selected for pandemic influenza vaccines worldwide is antigenically similar to nearly all isolates that have been examined to date.⁷ Multiple genetic groups have been recognized, including one recently predominant lineage,⁸ but any possible clinical importance of different lineages remains uncertain. Reassortment has not occurred with human influenza viruses to date. The level of pulmonary replication of the 2009 H1N1 virus has been higher than that of seasonal influenza A (H1N1) viruses in experimentally infected animals,^9,10,11 but the 2009 pandemic strain generally lacks mutations that are associated with increased pathogenicity in other influenza viruses (Table 1 in the Supplementary Appendix).

Epidemiology

Infection, Illness, and Disease Burden

Most illnesses caused by the 2009 H1N1 virus have been acute and self-limited, with the highest attack rates reported among children and young adults. The relative sparing of adults older than 60 years of age^3,12,13 is presumably due to the exposure of persons in this age group to antigenically related influenza viruses earlier in life, resulting in the development of cross-protective antibodies (Table 2 in the Supplementary Appendix).^10,14

Rates of illness from 2009 H1N1 virus infection have varied, but during one outbreak in New Zealand, the attack rate of illness was estimated at 7.5%, and the attack rate of overall infection was estimated at 11%.¹⁵ An estimated one third of infections in one boarding school were subclinical.¹⁶ After the peak of a second wave of infection in Pittsburgh, the seroprevalence of hemagglutination-inhibition antibody suggested that about 21% of all persons and 45% of those between the ages of 10 and 19 years had become infected.¹⁷

The overall case fatality rate has been less than 0.5%, and the wide range of estimates (0.0004 to 1.47%) reflects uncertainty regarding case ascertainment and the number of infections.^18,19,20 The case fatality rate for symptomatic illness was estimated to be 0.048% in the United States²¹ and 0.026% in the United Kingdom.¹³ In contrast to seasonal influenza, most of the serious illnesses caused by the pandemic virus have occurred among children and nonelderly adults, and approximately 90% of deaths have occurred in those under 65 years of age.

Rates of hospitalization and death have varied widely according to country.²² Hospitalization rates have been highest for children under the age of 5 years,²² especially those under the age of 1 year, and lowest for persons 65 years of age or older.²³ In the United States, among patients who were hospitalized with pandemic influenza, 32 to 45% were under the age of 18 years.^23,24 Approximately 9 to 31% of hospitalized patients have been admitted to an intensive care unit (ICU), where 14 to 46% of patients have died.^{23,24,25,26,27} The overall case fatality rate among hospitalized patients appears to have been highest among those 50 years of age or older and lowest among children.^1,13,23,27

Transmission and Outbreaks

The mechanisms of person-to-person transmission of the 2009 H1N1 virus appear to be similar to those of seasonal influenza, but the relative contributions of small-particle aerosols, large droplets, and fomites are uncertain. Rates of secondary outbreaks of illness vary according to the setting and the exposed population, but estimates range from 4 to 28%. Household transmission is highest among children and lowest among adults over 50 years of age.^28,29 In the United Kingdom and the United States, the rates of secondary outbreaks in households were 7% and 13%, respectively, with children at increased risk for infection by a factor of two to four.^16,28 Many outbreaks have occurred in schools, day-care facilities, camps, and hospitals.^16,30,31 Estimates of the basic reproduction number (the mean number of secondary cases of infection transmitted by a single primary case in a susceptible population) generally range from 1.3 to 1.7 according to the setting, which are similar to or slightly higher than the estimates for seasonal influenza,^20,32,33 but may be as high as 3.0 to 3.6 in outbreaks in crowded schools.³¹

Risk Groups and Risk Factors for Severe Disease

Approximately one quarter to one half of patients with 2009 H1N1 virus infection who were hospitalized or died had no reported coexisting medical conditions.^{13,23,26,27,34} Underlying conditions that are associated with complications from seasonal influenza also are risk factors for complications from 2009 H1N1 virus infection (Table 1). Pregnant women (especially those in the second or third trimester), women who are less than 2 weeks post partum, and patients with immunosuppression or neurologic disorders have also been overrepresented among those with severe 2009 H1N1 virus infection.^23,24,26,35 Although pregnant women represent only 1 to 2% of the population, among patients with 2009 H1N1 virus infection, they have accounted for up to 7 to 10% of hospitalized patients,^22,23,24 6 to 9% of ICU patients,^26,27 and 6 to 10% of patients who died.^23,35 There appears to be a particularly increased risk of death among infected women during the third trimester,³⁶ especially among those who have coinfection with the human immunodeficiency virus (HIV).³⁷

View this table: [in this window] [in a new window]   Table 1. Risk Factors for Complications of or Severe Illness with 2009 H1N1 Virus Infection.

 
Among patients with severe or fatal cases of 2009 H1N1 virus infection, severe obesity (body-mass index [the weight in kilograms divided by the square of the height in meters], 35) or morbid obesity (body-mass index, 40) has been reported at rates that are higher by a factor of 5 to 15 than the rate in the general population.^23,26,27,38 In addition to obesity-associated risks, such as cardiovascular disease and diabetes, possible adverse immunologic effects and management problems related to obesity may be contributory.

In certain disadvantaged groups, including indigenous populations of North America and the Australasia–Pacific region, rates of severe 2009 H1N1 virus infection have been increased by a factor of five to seven.^23,26,27 Factors that may contribute to this trend include crowding; an increased prevalence of underlying medical disorders, alcoholism, and smoking²⁷; delayed seeking of or access to care; and possibly unidentified genetic factors. Aboriginal status, the presence of coexisting conditions, and delayed receipt of antiviral therapy were independently associated with severe disease in one Canadian study.³⁹

Pathogenesis

Viral Replication

Studies of hemagglutinin-receptor binding indicate that the 2009 H1N1 virus is well adapted to mammalian hosts and binds to both 2,6-linked cellular receptors (as do seasonal influenza viruses) and 2,3-linked receptors,⁴⁰ which are present in the conjunctivae, distal airways, and alveolar pneumocytes. The 2009 H1N1 virus shows increased ex vivo replication in human bronchial epithelium at 33°C, as compared with a seasonal influenza virus,⁴¹ and is also characterized by increased replication and pathological changes in the lungs of nonhuman primates and increased replication in ex vivo human lung tissues.¹⁰ Such observations may help explain the ability of the virus to cause severe viral pneumonitis in humans.

In uncomplicated illness, nasopharyngeal viral RNA loads peak on the day of onset of symptoms and decline gradually afterward.⁴² However, viral replication may be more prolonged than in seasonal influenza, and on day 8 of uncomplicated illness in adults and teenagers, nasopharyngeal swabs have yielded viral RNA in 74% of patients and infectious virus in 13% of patients.^30,43 Infectious virus has been recovered from children up to 6 days after the resolution of fever.

Nasopharyngeal viral loads are increased in patients with severe pneumonia and decline slowly in critically ill patients.⁴⁴ Among intubated patients, viral RNA has been detected at higher levels and for longer periods in the lower respiratory tract than in the upper respiratory tract.⁴⁵ Viral RNA may be detected in secretions from the lower respiratory tract up to 28 days after the onset of severe pneumonia⁴⁶ and longer in patients with immunosuppression. Viral RNA and (infrequently) infectious virus have been detected in the stool of patients, and viral RNA has been detected infrequently in blood or urine of patients,^44,45 although one small study reported the frequent detection of viral RNA in blood, regardless of the severity of the illness.⁴⁷

Immune Responses

The patterns of innate and adaptive immune responses in patients with 2009 H1N1 virus infection are incompletely characterized. Seasonal and pandemic 2009 H1N1 viruses induce similar proinflammatory mediator responses in human cells in vitro⁴¹ but do not activate effective innate antiviral responses in human dendritic cells and macrophages.⁴⁸ Increased plasma levels of interleukin-15, interleukin-12p70, interleukin-8, and especially interleukin-6 may be markers of critical illness.^45,47 High systemic levels of interferon- and mediators involved in the development of type 1 and type 17 helper T-cell responses have been reported in hospitalized patients.⁴⁷ As compared with patients with less severe illness, patients who died or who had the acute respiratory distress syndrome (ARDS) had increased plasma levels of interleukin-6, interleukin-10, and interleukin-15 throughout the illness and of granulocyte colony-stimulating factor, interleukin-1, interleukin-8, interferon-inducible protein 10, and tumor necrosis factor during the late phase of illness.⁴⁴ Levels of serum hemagglutination-inhibition and neutralizing antibodies rise promptly after infection in immunocompetent persons,¹⁴ but symptomatic reinfections have been reported.⁴⁹

Pathological Features

In fatal cases of H1N1 virus infection, the most consistent histopathological findings are varying degrees of diffuse alveolar damage with hyaline membranes and septal edema, tracheitis, and necrotizing bronchiolitis^50,51,52 (Figure 1). Other early changes include pulmonary vascular congestion and, in some cases, alveolar hemorrhage. In addition to infecting cells in upper respiratory and tracheobronchial epithelium and mucosal glands, the 2009 H1N1 virus targets alveolar lining cells (type I and II pneumocytes)⁵⁰ (Figure 2). Viral antigens have been readily detectable in about two thirds of patients who died within 10 days after the onset of illness and may be detectable for more than 10 days.⁵⁰ Other autopsy findings include hemophagocytosis, pulmonary thromboemboli and hemorrhage, and myocarditis.⁴⁴ Bronchopneumonia with evidence of bacterial coinfection has been found in 26 to 38% of fatal cases.^50,51,52

View larger version (106K): [in this window] [in a new window]   Figure 1. Lung-Tissue Specimen Obtained at Autopsy from a 13-Year-Old Boy after a 7-Day Clinical Course of 2009 H1N1 Virus Infection. The specimen shows diffuse alveolar damage with hyaline membrane formation (arrows) and hemorrhage (hematoxylin and eosin). The patient, who had cerebral palsy, received oseltamivir for 2 days before he died. No evidence of bacterial coinfection was present. (Courtesy of Dr. Sherif R. Zaki, CDC.)

 

View larger version (102K): [in this window] [in a new window]   Figure 2. Immunostaining of Influenza Viral Antigens in Lung-Tissue Specimen Obtained at Autopsy from a 55-Year-Old Woman after a 7-Day Clinical Course of 2009 H1N1 Virus Infection. The specimen shows viral antigens (red color) in the nuclei of alveolar-lining cells (arrows), including type I and type II pneumocytes. Many infected cells have detached and are seen in alveolar spaces. Evidence of Streptococcus pneumoniae coinfection was also present in the patient, who had Down’s syndrome and hepatitis B infection (mouse anti-influenza nucleoprotein monoclonal antibody with naphthol fast-red substrate and hematoxylin counterstain). (Courtesy of Dr. Sherif R. Zaki, CDC.)

 
Clinical Features

Incubation Period

The incubation period appears to be approximately 1.5 to 3 days, which is similar to that of seasonal influenza.^{18,28,31,32,53} In a minority of patients, the period may extend to 7 days.

Clinical Presentation

Infection with the 2009 H1N1 virus causes a broad spectrum of clinical syndromes, ranging from afebrile upper respiratory illness to fulminant viral pneumonia. Mild illness without fever has been reported in 8 to 32% of infected persons.⁵³ Most patients presenting for care have typical influenza-like illness with fever and cough, symptoms that are sometimes accompanied by sore throat and rhinorrhea (Table 2).^{2,24,34,53,54,55,56} Systemic symptoms are frequent. Gastrointestinal symptoms (including nausea, vomiting, and diarrhea) occur more commonly than in seasonal influenza, especially in adults.^3,57 Dyspnea, tachypnea in children, chest pain, hemoptysis or purulent sputum, prolonged or recurrent fever, altered mental status, manifestations of dehydration, and reappearance of lower respiratory tract symptoms after improvement are signs of progression to more severe disease or complications.^{2,25,26,27,58}

View this table: [in this window] [in a new window]   Table 2. Symptom Profiles in Groups of Patients with Suspected or Confirmed Pandemic 2009 H1N1 Virus Infection Worldwide.

 
The principal clinical syndrome leading to hospitalization and intensive care is diffuse viral pneumonitis associated with severe hypoxemia, ARDS, and sometimes shock and renal failure.^26,27 This syndrome has accounted for approximately 49 to 72% of ICU admissions for 2009 H1N1 virus infection.^26,27 Rapid progression is common, typically starting on day 4 to 5 after the onset of illness, and intubation is often necessary within 24 hours after admission. Currently available prognostic algorithms for community-acquired pneumonia, such as CURB-65 (a measure of confusion, urea nitrogen, respiratory rate, and blood pressure and an age of 65 years or older), may not apply.⁵⁸ Radiographic findings commonly include diffuse mixed interstitial and alveolar infiltrates, although lobar and multilobar distributions occur, particularly in patients with bacterial coinfection. Chest computed tomography has shown multiple areas of ground-glass opacities, air bronchograms, and alveolar consolidation, particularly in the lower lobes.²⁴ Small pleural effusions occur, but an increased volume suggests volume overload or possibly empyema. Pulmonary thromboemboli have occurred in some critically ill patients with ARDS.

Other important syndromes include severe, prolonged exacerbation of chronic obstructive pulmonary disease (COPD) or asthma (in about 14 to 15% of patients), bacterial coinfections, and decompensation of serious coexisting conditions (Table 1).^23,26,27 Among hospitalized patients with 2009 H1N1 infection, a history of asthma has been reported in 24 to 50% of children and adults, and COPD in 36% of adults.^23,24 Bacterial pneumonia, usually caused by Staphylococcus aureus (often methicillin-resistant), Streptococcus pneumoniae, S. pyogenes, and sometimes other bacteria, has been suspected or diagnosed in 20 to 24% of ICU patients and has been found in 26 to 38% of patients who died, often in association with a short clinical course.^26,27,50,52 Death from 2009 H1N1 virus and bacterial coinfection has occurred within 2 to 3 days in some cases. Sporadic cases of neurologic manifestations (confusion, seizures, unconsciousness, acute or postinfectious encephalopathy, quadriparesis, and encephalitis)⁵⁹ and myocarditis have been reported, including some fulminant cases.

Laboratory findings at presentation in patients with severe disease typically include normal or low-normal leukocyte counts with lymphocytopenia and elevations in levels of serum aminotransferases, lactate dehydrogenase, creatine kinase, and creatinine.^2,25,27 Myositis and rhabdomyolysis have occurred in severe cases. A poor prognosis is associated with increased levels of creatine kinase, creatinine, and perhaps lactate dehydrogenase, as well as with the presence of thrombocytopenia and metabolic acidosis (Table 3 in the Supplementary Appendix).²

Special Populations

Young children with 2009 H1N1 virus infection may have marked irritability, severe lethargy, poor oral intake, dehydration resulting in shock, and seizures.^56,60 Other complications include invasive bacterial coinfections, encephalopathy or encephalitis (sometimes necrotizing), and diabetic ketoacidosis.^59,61 Bronchiolitis in infants and croup in young children may require hospitalization but do not usually necessitate ICU care. Suspected transplacental transmission of the 2009 H1N1 virus has been reported,⁶² and respiratory transmission from a symptomatic mother to a newborn can occur during the postpartum period. Newborn infants may also have apnea, tachypnea, cyanosis, and lethargy. Pregnant women are at increased risk for severe illness, spontaneous abortion, preterm labor and birth, and fetal distress.^35,36,57

Afebrile or atypical presentations have occurred in pregnant women, patients with immunosuppression, those undergoing hemodialysis, and other risk groups (Table 1). Patients with severe immunosuppression are at increased risk for protracted viral replication and pneumonia.^63,64

Diagnosis

Clinical Factors

Clinical suspicion and the accuracy of diagnosis vary substantially, depending on whether the case occurs sporadically or during a recognized outbreak, when a typical presentation of influenza-like illness is likely to represent 2009 H1N1 virus infection. However, the wide clinical spectrum of 2009 H1N1 virus infection and its features that overlap with those of other common infections have sometimes led to the misdiagnosis of other potentially treatable infections (e.g., legionellosis, meningococcemia, leptospirosis, dengue, and malaria).⁵⁸ Coinfection with dengue or certain respiratory viruses (parainfluenza virus and respiratory syncytial virus) and detection of S. pneumoniae have been reported in some patients with severe 2009 H1N1 virus infection.⁶⁵ Coinfection with other respiratory viruses, including seasonal influenza virus, has also been reported.^34,65

Virologic Factors

Viral RNA detection by conventional or real-time reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay remains the best method for the initial diagnosis of 2009 H1N1 virus infection.⁵⁸ Nasopharyngeal aspirates or swabs taken early after the onset of symptoms are suitable samples, but endotracheal or bronchoscopic aspirates have higher yields in patients with lower respiratory tract illness.^46,58,66 One study showed that among patients with detectable H1N1 viral RNA in bronchoscopic samples, 19% had negative upper respiratory tract samples.⁶⁶ Negative lower respiratory tract samples have been noted in 10% or more of patients with severe 2009 H1N1 virus infection. Consequently, negative results in single respiratory specimens do not rule out 2009 H1N1 virus infection, and repeated collection of multiple respiratory specimen types is recommended when clinical suspicion is high.

Commercially available rapid influenza antigen assays have poor clinical sensitivity (11 to 70%) for the detection of 2009 H1N1 virus in respiratory specimens and cannot differentiate among influenza A subtypes (Table 4 in the Supplementary Appendix). Consequently, negative test results should not be used to make decisions with respect to treatment or infection control. Direct or indirect immunofluorescence tests are less sensitive than RT-PCR.⁶⁶

The 2009 H1N1 virus replicates in various cell types,⁶⁷ but isolation usually takes several days. Serologic assays (microneutralization and hemagglutination inhibition) that detect increases in antibody levels in paired serum samples provide a retrospective diagnosis; single high titers in serum samples from convalescent patients may be indicative of recent infection,¹⁴ but routine testing of a single specimen to detect recent infection is not recommended.

Clinical Management

Antiviral Therapy

The currently circulating 2009 H1N1 virus is susceptible to the neuraminidase inhibitors oseltamivir (Tamiflu) and zanamivir (Relenza) but is almost always resistant to amantadine and rimantadine.^3,10 Therapy with a neuraminidase inhibitor is especially important for patients with underlying risk factors, including pregnancy,⁶⁸ and those with severe or progressive clinical illness (Table 3).⁶⁹ Standard doses of oseltamivir or inhaled zanamivir can be used for the treatment of mild illness, unless viral resistance to oseltamivir has been documented or is suspected (e.g., because of chemoprophylaxis failure), in which case zanamivir is preferred.

View this table: [in this window] [in a new window]   Table 3. Antiviral Therapy in Specific Subgroups of Patients.

 
Early therapy with oseltamivir in patients with 2009 H1N1 virus infection may reduce the duration of hospitalization⁷⁰ and the risk of progression to severe disease requiring ICU admission or resulting in death.^24,35,36 In one study involving 45 patients with 2009 H1N1 virus who had cancer or had undergone hematopoietic stem-cell transplantation, 18% had pneumonia and 37% were hospitalized; all patients received oseltamivir, and no deaths were reported.⁷¹ Oseltamivir-treated patients with HIV infection who were receiving highly active antiretroviral therapy had a clinical course similar to that in immunocompetent persons.⁷² Deaths have occurred despite early therapy,⁷³ but the administration of oseltamivir even after an interval of more than 48 hours since the onset of illness has been associated with reduced rates of death among hospitalized patients infected with the 2009 H1N1 virus,²⁵ seasonal influenza virus, or H5N1 virus. Decisions regarding antiviral treatment should not await laboratory confirmation, and patients presenting with progressive illness more than 48 hours after the onset of illness should be treated empirically with oseltamivir as soon as possible. Patients with progressive or severe illness who have a negative initial test result for 2009 H1N1 virus should continue to receive therapy unless an alternative diagnosis is established.

In uncomplicated illness, the early use of oseltamivir is usually associated with prompt clearance of infectious 2009 H1N1 virus from the upper respiratory tract.⁵³ However, infectious virus has commonly been detected after the resolution of fever and has sometimes been detected after the completion of therapy,³⁰ and viral RNA of uncertain clinical significance may be detectable for up to 12 days after the onset of illness.⁷⁴ In one study, the independent risk factors for prolonged viral RNA detection were an age of less than 14 years, male sex, and an interval of more than 48 hours between the onset of illness and the start of oseltamivir treatment.⁵³

In severely ill patients, viral RNA may be detectable in endotracheal aspirates for several weeks after the initiation of oseltamivir therapy.^45,46 An increased dose of the drug (e.g., 150 mg twice daily in adults) and particularly an increased duration of therapy (e.g., a total of 10 days) with avoidance of treatment interruptions are reasonable in patients with pneumonia or evidence of clinical progression.⁶⁹ Doses of up to 450 mg twice daily have been administered successfully in healthy adults, and controlled studies of higher-dose regimens are in progress. Higher weight-adjusted doses are also required in infants and young children to provide drug exposure similar to that in adults.^69,75 Bioavailability in critically ill patients receiving oseltamivir by nasogastric tube appears to be similar to that in patients with uncomplicated illness.⁷⁶ The tolerability and efficacy of inhaled zanamivir have not been adequately studied in patients with severe influenza. However, the failure of inhaled zanamivir therapy to clear virus in patients with pneumonia has been reported.⁶³ Some seriously ill patients treated with inhaled zanamivir have had respiratory distress, and nebulized delivery of extemporaneously prepared solutions of zanamivir powder with its lactose carrier has been associated with lethal ventilator dysfunction.⁷⁷

Oseltamivir Resistance

A His275Tyr mutation in viral neuraminidase confers high-level resistance to oseltamivir but not to zanamivir.^3,78 Most oseltamivir-resistant 2009 H1N1 viruses have been sporadic isolates from treated patients, particularly those with immunosuppression who received prolonged oseltamivir therapy^63,64 or those in whom postexposure oseltamivir chemoprophylaxis failed.⁷⁸ However, oseltamivir-resistant isolates have been found in patients without known exposure to oseltamivir and in limited clusters of cases associated with person-to-person transmission in otherwise healthy patients and those with immunosuppression.^78,79 Although in most cases oseltamivir-resistant variants have caused mild, self-limited illness, they have been associated with pneumonia in children and with severe, sometimes fatal illness in patients with immunosuppression.^64,78,80

Intravenous Neuraminidase Inhibitors

Intravenous administration of zanamivir or peramivir provides rapid drug delivery at high levels (Table 5 in the Supplementary Appendix). The efficacy of intravenous peramivir appeared to be similar to that of oseltamivir in one study of adults hospitalized with seasonal influenza,⁸¹ but peramivir is less active by a factor of at least 80 for oseltamivir-resistant viruses carrying the His275Tyr mutation than for oseltamivir-susceptible viruses. Intravenous zanamivir (if available) is the preferred option for hospitalized patients with suspected or documented oseltamivir-resistant 2009 H1N1 virus infection.^63,64,80 Both drugs are available on a compassionate-use basis for treating seriously ill patients, and peramivir was recently authorized for emergency use in hospitalized patients in the United States⁸¹ and licensed for use in Japan.

General principles of clinical management and prevention are summarized in WHO⁵⁸ and country-specific guidelines and are reviewed in the Supplementary Appendix.

Future Directions

A large amount of information about the natural history and clinical management of 2009 H1N1 virus infection has been obtained in a remarkably short period of time, but considerable gaps remain. The uncertain evolution of this virus among humans and potentially other species highlights the need for continued virologic surveillance for antigenic changes, viral reassortment, antiviral resistance, and altered virulence. Improvements in the global capacity for detection of influenza viruses by molecular analysis, such as RT-PCR assay, and by viral isolation are needed. A simple, inexpensive, highly accurate rapid influenza diagnostic test that is easily deployable worldwide has yet to be developed. The burden and character of disease in low-resource settings are still incompletely understood,⁸² especially with respect to disadvantaged populations, including marginalized, refugee, and aboriginal populations. Poverty, homelessness, illiteracy, recent immigration, language barriers, and cultural factors may impede access to care, with the potential for more serious outcomes of influenza. Thus, public health efforts reduce risk factors and to identify at-risk populations for the purpose of providing immunization and early care, including the use of antiviral drugs, should focus on social as well as clinical factors. Both experience with previous pandemics and recent modeling efforts indicate that the age bias observed for outbreaks of 2009 H1N1 virus infection may shift in coming months toward older persons, with implications for the allocation of public health resources.⁸³

Major gaps exist in our understanding of viral transmission, pathogenesis of disease, genetic and other host factors related to susceptibility^84,85 or disease severity, and optimal management of severe illness. The development of new antiviral regimens with improved effectiveness, combinations with targeted adjunctive therapies (i.e., immunodulators and neutralizing antibodies or immunotherapy), and improved management of influenza-associated ARDS are priorities, along with better prevention, recognition, and treatment of invasive bacterial coinfections. Available findings highlight the importance of early use of antiviral drugs and antibiotics in the treatment of serious cases and of the potential value of influenza-specific and pneumococcal vaccines for prevention. Both the gaps in knowledge and the experience to date underline the urgent need for better international collaboration in clinical research, particularly in the case of diseases with pandemic potential, for which rapid detection, investigation, and characterization of clinical syndromes are prerequisites for improved mitigation of their public health consequences.

Dr. Kumar reports receiving grant support (to the University of Manitoba) from Roche for studies of oseltamivir; and Dr. Nicholson, receiving travel expenses and lecture and consulting fees from Baxter, Novartis, and GlaxoSmithKline. No other potential conflict of interest relevant to this article was reported.

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

The opinions expressed in this article are those of the members of the Writing Committee and do not necessarily reflect those of the institutions or organizations with which they are affiliated.

We thank Rebecca Harris of the WHO, Geneva, for her assistance in the preparation of the manuscript and our colleagues who shared unpublished information during the WHO Consultation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza, October 14–16, 2009, in Washington, D.C., and subsequently.

^* The members of the Writing Committee of the World Health Organization (WHO) Consultation on Clinical Aspects of Pandemic (H1N1) 2009 Influenza, who are listed in the Appendix, assume responsibility for the content of the article.

Address reprint requests to Dr. Frederick G. Hayden at P.O. Box 800473, University of Virginia Health System, Charlottesville, VA 22908, or at fgh@virginia.edu

.

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Appendix

The members of the Writing Committee are as follows: Edgar Bautista, M.D., National Institute of Respiratory Diseases, Mexico City; Tawee Chotpitayasunondh, M.D., Queen Sirikit National Institute of Child Health, Bangkok, Thailand; Zhancheng Gao, M.D., Ph.D., Peking University People’s Hospital, Beijing; Scott A. Harper, M.D., M.P.H., Michael Shaw, Ph.D., Timothy M. Uyeki, M.D., M.P.H., M.P.P. (coeditor), and Sherif R. Zaki, M.D., Ph.D., Centers for Disease Control and Prevention, Atlanta; Frederick G. Hayden, M.D. (editor), University of Virginia, Charlottesville, and Wellcome Trust, London; David S. Hui, M.D., Chinese University of Hong Kong, Hong Kong; Joel D. Kettner, M.D., University of Manitoba and Manitoba Health, and Anand Kumar, M.D., University of Manitoba — both in Winnipeg, Canada; Matthew Lim, M.D., Nahoko Shindo, M.D., Ph.D., and Charles Penn, Ph.D., World Health Organization, Geneva; and Karl G. Nicholson, M.D., University of Leicester, Leicester, United Kingdom.

2009 신종플루 바이러스의 특이항원에만 결합할 수 있는  MF59 보조제를 첨가한 백신접종에 대한 결과가 [ N Engl J Med ] 최신호에 보고되었습니다.(지난 10월에 preliminary version이 출판된 바 있습니다)

통증, 근육 통증, 발열 등의 증상이 조금 더 나타나긴 했지만 전체적으로 보조제를 첨가하지 않은 백신보다 항체가가 높게 나왔다고 합니다. 

이 연구가 노바티스사로부터 후원을 받은 것이라는 점도 고려해서 읽어야 할 것 같구요… 저자들은 Novartis Vaccines and Sanofi Pasteur로부터 자문료를 받았고… Baxter Vaccines and Novartis Vaccines로부터 강의료를 받았으며… Hoffmann–La Roche, Novartis Vaccines, and GlaxoSmithKline로부터 지원을 받았음을 밝혔습니다.

================================

 2009 Influenza A (H1N1) Monovalent MF59-Adjuvanted Vaccine

Tristan W. Clark, M.R.C.P., Manish Pareek, M.R.C.P., Katja Hoschler, Ph.D., Helen Dillon, M.R.C.P., Karl G. Nicholson, M.D., F.R.C.P., Nicola Groth, M.D., and Iain Stephenson, M.D., F.R.C.P.

출처 : [ N Engl J Med ] Volume 361:2424-2435  December 17, 2009  Number 25
http://content.nejm.org/cgi/content/full/361/25/2424

ABSTRACT

Background The 2009 pandemic influenza A (H1N1) virus has emerged to cause the first pandemic of the 21st century. Development of effective vaccines is a public health priority.

Methods We conducted a single-center study, involving 176 adults, 18 to 50 years of age, to test the monovalent influenza A/California/2009 (H1N1) surface-antigen vaccine, in both MF59-adjuvanted and nonadjuvanted forms. Subjects were randomly assigned to receive two intramuscular injections of vaccine containing 7.5 µg of hemagglutinin on day 0 in each arm or one injection on day 0 and the other on day 7, 14, or 21; or two 3.75-µg doses of MF59-adjuvanted vaccine, or 7.5 or 15 µg of nonadjuvanted vaccine, administered 21 days apart. Antibody responses were measured by means of hemagglutination-inhibition assay and a microneutralization assay on days 0, 14, 21, and 42 after injection of the first dose.

Results The most frequent local and systemic reactions were pain at the injection site and muscle aches, noted in 70% and 42% of subjects, respectively; reactions were more common with the MF59-adjuvanted vaccine than with nonadjuvanted vaccine. Three subjects reported fever, with a temperature of 38°C or higher, after either dose. Antibody titers, expressed as geometric means, were higher at day 21 among subjects who had received one dose of MF59-adjuvanted vaccine than among those who had received one dose of nonadjuvanted vaccine (P<0.001 by the microneutralization assay). By day 21, hemagglutination-inhibition and microneutralization antibody titers of 1:40 or more were seen in 77 to 96% and 92 to 100% of subjects receiving MF59-adjuvanted vaccine, respectively, and in 63 to 72% and 67 to 76% of those receiving nonadjuvanted vaccine, respectively. By day 42, after two doses of vaccine, hemagglutination-inhibition and microneutralization antibody titers of 1:40 or more were seen in 92 to 100% and 100% of recipients of MF59-adjuvanted vaccine, respectively, and in 74 to 79% and 78 to 83% of recipients of nonadjuvanted vaccine, respectively.

Conclusions Monovalent 2009 influenza A (H1N1) MF59-adjuvanted vaccine generates antibody responses likely to be associated with protection after a single dose is administered. (ClinicalTrials.gov number, NCT00943358 [ClinicalTrials.gov] .)

Traditional seasonal influenza vaccines are produced from reassortant vaccine strains grown in hens’ eggs. However, demand for vaccine against the 2009 H1N1 virus will most likely exceed the supply if this method of manufacturing is solely used. Cell culture provides an additional platform for the manufacture of vaccines that may be more easily scaled up during periods of heightened demand.^5,6,7

Serologic analysis suggests that after seasonal vaccination in children and young adults, there is little evidence of cross-reactive antibodies against the 2009 H1N1 virus,⁸ with no evidence of protection from the seasonal vaccine.⁹ The efficacy of conventional vaccines prepared from avian influenza strains is disappointingly low, even after two doses.^{10,11,12,13,14} The addition of oil-in-water–emulsion adjuvant enhances immunogenicity and induces cross-reactive antibodies against antigenically drifted variants.^{12,13,14,15,16} The use of such adjuvants in 2009 influenza A (H1N1) vaccines has been suggested by the World Health Organization.¹⁷

Vaccination programs for 2009 influenza A (H1N1) are under way, but the optimal formulation is unknown. The need for high-yield vaccine strains, limitations of the supply and production capacity of egg-based vaccines, and the possible requirement of two doses in some groups may delay an effective immunization program.

We present the clinical and immunogenicity profiles of the 7.5-µg dose of the monovalent influenza A/California/2009 (H1N1) MF59-adjuvanted surface-antigen vaccine, derived from cell culture, administered to adults 18 to 50 years of age. Two doses of 7.5 µg of MF59-adjuvanted vaccine were given concurrently on day 0 or were given 7, 14, or 21 days apart; or two doses of 3.75 µg of MF59-adjuvanted vaccine or 7.5 or 15 µg of nonadjuvanted vaccine were given 21 days apart. Our earlier report of the preliminary results is available at NEJM.org.

Methods

The study was designed by one academic author and one industry author; the academic author was responsible for managing the data and drafting the manuscript. The data were fully accessible and interpreted by all the authors, who vouch for the accuracy and completeness of the data and analyses. The U.K. Medicines and Healthcare Products Regulatory Agency and the Leicestershire, Rutland, and Northamptonshire Ethics Committee approved the study. University Hospitals Leicester was the main sponsor; the vaccine was manufactured by Novartis, who provided funding but had no role in the conduct of the study or in preparation of the manuscript.

Vaccine

The 2009 H1N1 vaccine virus (New York Medical College [NYMC] X-179A) was generated from the influenza A/California/7/2009 strain with the use of classical reassortant methods. The gene segments encoding the hemagglutinin, neuraminidase, and the polymerase PB1 were derived from the influenza A/California/7/2009 strain, with the remaining genes taken from the influenza A/PR8/8/34 virus used as a backbone for influenza vaccines. The strain was supplied by the Centers for Disease Control and Prevention and is a pandemic vaccine strain recommended for use in vaccine development. The seed virus was grown in Madin–Darby Canine Kidney (MDCK) cell culture by means of standard processes similar to those used for the development of Optaflu vaccines against interpandemic influenza. The vaccine was formulated and produced by Novartis (Marburg, Germany) as an inactivated surface-antigen H1N1 vaccine, with or without MF59 adjuvant, and was supplied in 0.5-ml prefilled single-dose syringes. Each MF59-adjuvanted vaccine contained 7.5 µg of H1 hemagglutinin, 9.75 mg of the squalene MF59, 1.175 mg of polysorbate 80, and 1.175 mg of sorbitan trioleate in buffer. Each nonadjuvanted vaccine contained 15 µg of H1 hemagglutinin in buffer. Hemagglutinin content in the final vaccine was initially determined by means of reverse-phase high-performance liquid chromatography, because single-radial diffusion reagents were unavailable. Subsequently, hemagglutinin content of the final product, determined by means of single-radial diffusion reagents, was approximately 20% lower than the estimated content. Vaccine was stored at 2 to 8°C until use.

Study Design

We conducted a single-center, phase 1, randomized study from July through September 2009 at Leicester Royal Infirmary (Leicester, United Kingdom). Subjects were screened for eligibility and provided written informed consent. (For eligibility criteria, see the Supplementary Appendix, available with the full text of this article at NEJM.org.)

The first 75 subjects enrolled were randomly assigned, in a 1:1:1 ratio, to receive two doses of 7.5 µg of MF59-adjuvanted vaccine, either concurrently administered on day 0 (i.e., one injection of the vaccine containing twice the antigen and adjuvant content of a single vaccine) or administered in two doses, one at day 0 and the other at day 7, 14, or 21. Serum samples for antibody measurements were collected on days 0, 14, 21, and 28.

The next 101 subjects enrolled were randomly assigned, in a 1:1:1:1 ratio, to receive two doses of 7.5 or 3.75 µg of MF59-adjuvanted vaccine (for the latter dose, by administering half the contents of the adjuvanted-vaccine syringe for each), two 15-µg doses of nonadjuvanted vaccine, or two 7.5-µg doses of nonadjuvanted vaccine (by administering half the contents of the nonadjuvanted-vaccine syringe for each) — with one injection at day 0 and the other at day 21. Serum samples were collected on days 0, 14, 21, and 42.

The vaccine was administered by intramuscular injection into the deltoid muscle of the nondominant arm, or in both arms if both doses were given on day 0. Subjects were observed for 30 minutes after each injection, and for the next 7 days they recorded, in self-completed diaries, the severity of unsolicited and solicited local symptoms (pain, bruising, erythema, and swelling) and systemic symptoms (chills, malaise, muscle aches, nausea, and headache), oral temperature, and use of analgesics. Symptoms were graded as follows: none; mild, if they did not interfere with normal activities; moderate, if they resulted in interference with normal activities; and severe, if they prevented engagement in daily activities and necessitated medical attention. Adverse reactions were defined as any reaction that persisted beyond 7 days after vaccination. Serious adverse reactions were defined as any reaction that necessitated medical attention or hospitalization during the study period.

Laboratory Assays

Antibody responses were detected by means of microneutralization and hemagglutination-inhibition assays, according to standard methods,^18,19 at the Centre for Infections, Health Protection Agency (London), and with the use of cell-culture X-179A H1N1 vaccine virus (see the Vaccine section above) and egg-grown NIBRG-121 virus — a reverse-genetic virus containing hemagglutinin and neuraminidase from the influenza A/California/7/2009 strain (see the Supplementary Appendix for details). Serum samples obtained from subjects were tested with the use of 1:2 serial dilutions. For hemagglutination-inhibition assays, serum samples were tested at an initial dilution of 1:8, and those that were negative for the antibody were assigned a titer of 1:4. Serum specimens were analyzed to determine absolute end-point titers. For microneutralization assays, serum samples were tested at an initial dilution of 1:10,²⁰ and those that were negative were assigned a titer of 1:5. The final dilution was 1:320, and samples for which the end-point titers were greater were assigned a value of 1:640. Specimens were tested in duplicate, and the geometric mean values were used in analyses.

Statistical Analysis

The group sizes used are usual for phase 1 studies and were not based on power calculations. Data analysis was undertaken with the use of Stata software (version 9.2, StataCorp).

For solicited and unsolicited adverse reactions, the percentages of subjects (point estimates and 95% confidence intervals) with postvaccination reactions were based on the frequency and severity of the reported responses after vaccination. Exact (Clopper–Pearson) confidence intervals are reported for all proportional end points. We used a two-sided Fisher’s exact test to compare proportions between vaccine groups. All reported P values are two-sided, with no adjustment for multiple testing; values of 0.05 or less were considered to indicate statistical significance.

For immunogenicity analyses, the geometric mean antibody titers at each time point were used. Geometric mean titers and 95% confidence intervals were computed by taking the exponent (log₁₀) of the mean and of the lower and upper limits of the 95% confidence intervals of the log₁₀-transformed titers. Geometric mean titers were compared between each pair of vaccine groups by means of one-way analysis of variance on the log₁₀-transformed titers with Bonferroni correction for multiple pairwise comparisons, if appropriate. The proportions of subjects in whom seroconversion (a prevaccination hemagglutination-inhibition antibody titer 1:10 and a postvaccination titer 1:40 or a prevaccination titer 1:10 and an increase in the titer by a factor of four or more) or a hemagglutination-inhibition antibody titer of 1:40 or more was achieved were compared between each group with the use of a two-sided Fisher’s exact test. Separate analyses were performed for the hemagglutination-inhibition and microneutralization assays. Because there are no established immune correlates for microneutralization, in that analysis we assessed the proportion of subjects who had seroconversion (an increase in the antibody titer by a factor of four or more) and a microneutralization titer of 1:40 or more.

Results

We enrolled 176 subjects. A total of 101 subjects received two 7.5-µg doses of MF59-adjuvanted vaccine (with the two doses administered concurrently at day 0 [in 25 subjects] or one dose at day 0 and one at day 7 [in 25 subjects], at day 14 [in 25 subjects], or at day 21 [in 26 subjects]). The other 75 subjects each received two doses of 3.75 µg of MF59-adjuvanted vaccine (25 subjects), or 7.5 or 15 µg of nonadjuvanted vaccine (25 subjects each), separated by 21 days (i.e., doses at day 0 and at day 21).

Both doses of vaccine were given in 175 of the 176 subjects (99%); 1 subject in the group receiving 15 µg of nonadjuvanted vaccine did not attend the second vaccination visit. In all, 322 of the 325 issued diary cards (99%) were returned. Serum samples were obtained from 175 of the 176 subjects (99%) and 97 of the 101 subjects (96%) from whom samples were required at days 21 and day 42, respectively, according to protocol. In addition, serum samples were obtained on day 14 from 166 of the 176 subjects (94%). Data from all 176 subjects were included in safety and immunogenicity analyses (see the Supplementary Appendix).

The median age was 33 years (range, 18 to 50); 65% of the subjects were women, 82% were white, and 37% had previously received seasonal influenza vaccine (Table 1). The baseline characteristics were similar among the seven groups.

View this table: [in this window] [in a new window]   Table 1. Baseline Characteristics of the Study Subjects, According to Vaccine Group.

 
Safety Analysis

Solicited local and systemic reactions during the first 7 days following any vaccine dose are shown in Table 2. Overall, 80% of subjects reported adverse reactions after either vaccine dose (73% after the first and 60% after the second). The frequency or severity of reactions did not increase after the second dose was received (see the Supplementary Appendix). All self-reported reactions were graded as mild or moderate and were generally self-limited, resolving within a 72-hour period. No dose–response relationship was observed for either vaccine type for any reaction.

View this table: [in this window] [in a new window]   Table 2. Solicited Local and Systemic Adverse Effects within 7 Days after Receipt of Either Dose of MF59-Adjuvanted or Nonadjuvanted Vaccine, According to Vaccine Group.

 
The most frequent local reaction after any vaccine was pain at the injection site, reported by 61% of subjects. Overall, pain was more frequent after injection of the MF59-adjuvanted vaccine than with nonadjuvanted vaccine (65% vs. 39%, P=0.003). In general, pain was not accompanied by redness, swelling, or bruising, although bruising was reported to be severe in one subject — affecting an area 20 mm in diameter — after the first dose, with resolution within 72 hours. No severe local reactions were reported.

The most frequent systemic reaction was muscle ache, reported by 40% of subjects. There was no significant difference in frequency or severity of systemic reactions after receipt of MF59-adjuvanted vaccine and those after receipt of nonadjuvanted vaccine, except in subjects who received two doses of MF59-adjuvanted vaccine on day 0, who reported a greater frequency of muscle ache than did subjects who received one dose of MF59-adjuvanted vaccine on day 0 (P=0.02). A total of 13% of subjects reported use of analgesics. Three subjects reported fever, defined as a temperature of 38°C or more (after the first dose in two subjects and after the second dose in the third), but none required antipyretic medication. No severe systemic reactions were reported.

Fifteen unsolicited adverse events were reported (see the Supplementary Appendix). After receiving MF59-adjuvanted vaccine, three subjects reported self-limiting diarrhea (that resolved within the 48-hour period after the first dose); one subject took over-the-counter loperamide. Three subjects reported coryza that resolved within 72 hours. One subject reported toothache that resolved after 5 days. One subject reported a transient itchy rash on the right forearm that resolved within 48 hours. After receiving nonadjuvanted vaccine, two subjects reported musculoskeletal pain that resolved after 48 hours. Two subjects reported coryza that resolved within 72 hours. One subject each reported diarrhea, itching, or sore throat that resolved within 48 hours.

A probable vaccine-related adverse reaction, reported after receipt of the 7.5 µg of MF59-adjuvanted vaccine, is described in the Supplementary Appendix. Briefly, this subject (who received two doses of the MF59-adjuvanted vaccine on day 0) reported a purpuric rash on the lower limbs on day 17, with resolution within 72 hours. Further questioning revealed that she had consulted with her family practitioner in May 2009 for an intermittent leg rash within the 12-month period before the study. Investigations including complete blood count and biochemical profile showed normal values, but an autoimmune profile was positive for antinuclear and extractable nuclear-antigen antibodies. She had not received medication, and this medical history was not known at enrollment. Follow-up for the rash included a normal complete blood count and biochemical profile. Results of autoimmune testing were unchanged from those in May 2009.

Immunogenicity against the 2009 H1N1 Virus

Antibody responses against the vaccine strain were detected by the hemagglutination-inhibition assay (titer >1:8) and the microneutralization assay (titer >1:10) before vaccination in 16% and 31% of subjects, respectively; this frequency was unrelated to age (P=0.72 by hemagglutination-inhibition assay and P=0.32 by microneutralization assay) or previous receipt of seasonal vaccine (P=0.14 and P=0.18, respectively).

There was no significant dose–response relationship regarding the geometric mean titers, at any postvaccination visit, for MF59-adjuvanted vaccine (P=0.71 by hemagglutination-inhibition assay and P=0.43 by microneutralization assay on day 14; P=0.63 and P=0.42, respectively, on day 21; and P=0.86 and P=0.75, respectively, on day 42) or for nonadjuvanted vaccine (P=0.74 and P=0.49, respectively, on day 14; P=0.99 and P=0.62, respectively, on day 21; and P=0.27 and P=0.88, respectively, on day 42).

On day 14, geometric mean titers, as measured with the use of hemagglutination-inhibition assay (Table 3) and microneutralization assay (Table 4), were higher in subjects who received two 7.5-µg doses of MF59-adjuvanted vaccine by that time, as compared with those who had received one dose only (P=0.03 by hemagglutination-inhibition assay and P<0.001 by microneutralization assay). After the administration of one dose, the microneutralization antibody titers were greater in subjects who had received the MF59-adjuvanted vaccine than in those who had received nonadjuvanted vaccine (P<0.001).

View this table: [in this window] [in a new window]   Table 3. Antibody Responses as Measured with the Hemagglutination-Inhibition Assay, According to Vaccine Group.

 

View this table: [in this window] [in a new window]   Table 4. Antibody Responses as Measured with the Microneutralization Assay, According to Vaccine Group.

 
On day 21, microneutralization antibody titers were higher among subjects who had received two 7.5-µg doses of MF59-adjuvanted vaccine than among those who had received one dose only by that time (P=0.03). After one dose of either vaccine, the MF59-adjuvanted vaccine induced greater titers than nonadjuvanted vaccine (P<0.001 by microneutralization assay).

On day 42, after two doses of either vaccine, geometric mean titers were higher in groups receiving the MF59-adjuvanted vaccine than in those receiving the nonadjuvanted vaccine (P=0.007 by hemagglutination-inhibition assay and P<0.001 by microneutralization assay).

Table 3 shows the ratio of the antibody titer measured at each postvaccination visit and the titer measured at the prevaccination visit, and the percentages of subjects with seroconversion and with an antibody titer of 1:40 or more, as measured with the hemagglutination-inhibition assay.

There was no significant dose–response relationship regarding the rates of seroconversion with MF59-adjuvanted vaccine (P=0.78 by the hemagglutination-inhibition assay and P=1.00 by the microneutralization assay on day 14; P=0.29 and P=1.00, respectively, on day 21; and P=1.00 and P=1.00, respectively, on day 42) or with nonadjuvanted vaccine (P=0.23 and P=1.00, respectively, on day 14; P=0.23 and P=1.00, respectively, on day 21; and P=0.74 and P=1.00, respectively, on day 42). There was no significant difference in the rates of seroconversion between subjects who had a detectable prevaccination antibody titer and those who did not (day 14, P=0.46; day 21, P=0.61; and day 42, P=1.00). On day 14, the percentages of subjects with seroconversion and with an antibody titer of 1:40 or more were higher (P=0.007 and P=0.002, respectively) among subjects who had received two doses of the MF59-adjuvanted vaccine than among those who had received only one. On day 21, as compared with subjects who had received one dose of MF59-adjuvanted vaccine, those who had received two doses did not differ significantly in the percentage of subjects with seroconversion (P=0.06) but did have a greater percentage with an antibody titer of 1:40 or more (P=0.03). Although MF59-adjuvanted vaccine induced more seroconversions and antibody titers of 1:40 or more than nonadjuvanted vaccine at each postvaccination visit, the difference was not significant (day 14, P=0.22 and P=0.09, respectively; and day 21, P=0.07 and P=0.06, respectively). On day 42, after two vaccine doses, the percentages of subjects with seroconversion and an antibody titer of 1:40 or more were higher among the MF59-adjuvanted vaccine groups (P=0.05 and P=0.007, respectively) than among the nonadjuvanted vaccine groups.

Table 4 shows the ratio of the antibody titer measured at each postvaccination visit and the titer measured at the prevaccination visit, and the percentages of subjects with seroconversion and antibody titers of 1:40 or more, as measured with the microneutralization assay. On days 14 and 21, there were no significant differences between subjects who had received two doses of MF59-adjuvanted vaccine and those who had received one dose in the rate of seroconversion (day 14, P=0.20; and day 21, P=0.64) or in the percentage with titers of 1:40 or more (day 14, P=0.50; and day 21, P=1.00). On each postvaccination visit, the rate of seroconversion and the percentage of subjects with titers of 1:40 or more were greater after receipt of MF59-adjuvanted vaccine than after receipt of nonadjuvanted vaccine (day 14, P=0.02 and P=0.003, respectively; day 21, P=0.002 and P=0.001, respectively; and day 42, P=0.04 and P=0.001, respectively).

Figure 1 shows the distribution of antibody titers at day 14 and day 42, according to vaccine group. At day 14, hemagglutination-inhibition titers of 1:32 or more were achieved in 84% of subjects receiving MF59-adjuvanted vaccine and in 77% of subjects receiving nonadjuvanted vaccine. Microneutralization titers of 1:40 or more were achieved in 94% subjects receiving MF59-adjuvanted vaccine and in 73% of subjects receiving nonadjuvanted vaccine. After two doses, at day 42, hemagglutination-inhibition titers of 1:32 or more were achieved in 100% of subjects receiving MF59-adjuvanted vaccine and in 87% of subjects receiving nonadjuvanted vaccine. Microneutralization titers of 1:40 or more were achieved in 100% subjects receiving MF59-adjuvanted vaccine and in 80% of subjects receiving nonadjuvanted vaccine.

View larger version (27K): [in this window] [in a new window]   Figure 1. Reverse Cumulative-Distribution Curves of Antibody Titers in Serum Samples Obtained on Days 14 and 42, According to Number and Timing of Vaccine Doses. The percentages of subjects are based on the total number of subjects tested in each of the vaccine groups. All vaccine groups received the first of two doses on day 0. One group received the second dose of 7.5 µg of MF59-adjuvanted vaccine on day 0, one group on day 7, and one group on day 14; the three other groups received the second vaccine dose on day 21. Hemagglutination-inhibition titers (expressed as the reciprocal of the dilution) are shown at day 14 (Panel A) and day 42 (Panel B). Microneutralization titers are shown at day 14 (Panel C) and day 42 (Panel D). Titers are expressed as reciprocal of the dilution and are given on a log2 scale.

 
Responses against the NIBRG-121 virus were similar to those against the 2009 X-179A H1N1 vaccine virus, as measured by means of the hemagglutination-inhibition assay (see the Supplementary Appendix).

Discussion

Data from studies of other inactivated influenza vaccines suggest that hemagglutination-inhibition antibody titers of 1:40 or more provide partial protection, and this titer was achieved by most of the subjects given one dose, with or without adjuvant, in this clinical trial. Effective vaccination should reduce illness and virus transmission,⁴ although this may be challenging, as global demand for vaccine will probably exceed manufacturing capacity and will be met only by implementing a range of production approaches. Large-scale vaccine production with newly characterized viruses can be challenging if low egg growth limits the supply of antigen. Our vaccine was produced from a classical egg-derived seed virus propagated in a MDCK cell line.^7,21 Cell-culture systems may provide a faster response and greater scale-up than egg production. Cell-culture seasonal influenza seed viruses also show better antigenic matching to clinical isolates than egg-passaged strains.^5,6,7,8,21

Clinical experience with avian and human influenza A/H1N1–subunit vaccines in subjects who did not have detectable levels of preexisting antibody suggests that two doses are required to induce a hemagglutination-inhibition antibody titer of 1:40 or more.^{10,11,12,13,14,15,22,23} Traditionally, dosing intervals of 21 to 28 days are used, often delaying effective immunization. We evaluated rapid immunization schedules involving two doses of MF59-adjuvanted vaccines, since flexible dosing would be useful for authorities organizing immunizations. However, our data suggest that a single immunization against the 2009 pandemic influenza A (H1N1) virus would be sufficient to induce a hemagglutination-inhibition antibody titer of 1:40 or more.

Our findings add to observations that oil-in-water–emulsion adjuvants are well tolerated, with systemic reactogenicity similar to that of nonadjuvanted inactivated seasonal vaccines, but are associated with increased local pain at the site of administration.²⁴ For avian subvirion vaccines, oil-in-water–emulsion adjuvants are important to induce long-lasting cross-reactive immunity.^{10,12,13,14,15} Although 2009 pandemic influenza A (H1N1) isolates are antigenically homogenous, induction of broadly cross-reactive antibodies would be a desirable characteristic of the first vaccines against the 2009 H1N1 virus, and serum samples obtained after the administration of candidate vaccines, either adjuvanted or nonadjuvanted, should be assessed against emerging antigenic variant strains. The addition of MF59 adjuvant to 2009 influenza A (H1N1) monovalent vaccine increases the speed and magnitude of the antibody response; however, nonadjuvanted vaccine also induced satisfactory immune responses, which is consistent with early reports of other 2009 H1N1 subunit vaccines.²⁵ The 2009 H1N1 virus is antigenically distinct from recently circulating seasonal H1N1 strains, so the response to a single dose of vaccine suggests there may a greater degree of preexisting immunity in the population than expected. Sixteen percent of subjects had detectable prevaccination levels of hemagglutination-inhibition antibody, a finding that is consistent with results of seroepidemiologic studies.¹¹ Although we excluded subjects with previous respiratory illnesses, asymptomatic infection with influenza A (H1N1) viruses cannot be ruled out, since local activity was present during the study.

Interpretation of immunogenicity data for the vaccine against the 2009 pandemic influenza A (H1N1) virus is complicated by a lack of recognized immune correlates. The insensitivity of hemagglutination-inhibition assays to some avian hemagglutinin has required that microneutralization assays, hemagglutination-inhibition assays involving horse erythrocytes, or single radial hemolysis be used.^10,18,26 Because there is significant laboratory variation in testing,²⁰ efforts to develop biologic standards for serologic assays of influenza A (H1N1) viruses are under way.

The safety and immunogenicity of these and alternative candidate vaccines against the 2009 H1N1 virus, including egg-derived, whole-virion, recombinant, and live-attenuated vaccines, must be assessed in high-risk populations, including children, the elderly, and other persons whose immunologic profiles may differ from those of young adults.⁸ In addition, the duration of antibody responses and their ability to be boosted after revaccination should be established to predict protection against future pandemic waves.

Finally, although seasonal influenza vaccines have an established safety profile, there are occasional case reports of unusual reactions, including vasculitis.^24,27 MF59, a proprietary oil-in-water–emulsion adjuvant, was first licensed for use in seasonal influenza vaccines in 1997. Over 40 million doses have been delivered in Europe, and over 16,000 doses administered in clinical trials, with no excess reports of autoimmune conditions.²⁸ It is important to ensure post-marketing surveillance during any mass use of a pandemic-virus vaccine, with or without adjuvant. These results may be useful for planning of immunization schedules, and comparison with other vaccine options as they become available.

Supported by grants from University Hospitals Leicester and Novartis Vaccines and Diagnostics.

Dr. Hoschler reports holding equity and stock options in Illumina; Dr. Nicholson, receiving consulting fees from Novartis Vaccines and GlaxoSmithKline and lecture fees from Baxter Vaccines; Dr. Groth, holding equity and stock options in Novartis; and Dr. Stephenson, receiving consulting fees from Novartis Vaccines and Sanofi Pasteur, lecture fees from Baxter Vaccines and Novartis Vaccines, and grant support from Hoffmann–La Roche, Novartis Vaccines, and GlaxoSmithKline. No other potential conflict of interest relevant to this article was reported.

We thank the subjects who volunteered for the study; staff at University Hospitals of Leicester Infectious Diseases Research Unit, in particular, Sally Batham and Marie-jo Medina; Carolyn Maloney of the Department for Research and Development; Chris Cannaby of the Leicestershire Comprehensive Research Network Office; and the technical staff at the Health Protection Agency influenza laboratory for their assistance with serologic assessment.

Source Information

From the Infectious Diseases Unit, University Hospitals Leicester and Department of Inflammation, Infection and Immunity, University of Leicester, Leicester, United Kingdom (T.W.C., M.P., H.D., K.G.N., I.S.); the Respiratory Virus Laboratory, Health Protection Agency, London (K.H.); and Novartis Vaccines and Diagnostics, Siena, Italy (N.G.).

Drs. Clark and Pareek contributed equally to this article.

A preliminary version of this article (10.1056/NEJMoa0907650) was published on September 10, 2009, at NEJM.org.

Address reprint requests to Dr. Stephenson at the Infectious Diseases Unit, Level 6, Windsor Bldg., Leicester Royal Infirmary, Leicester, United Kingdom, or at iain.stephenson@uhl-tr.nhs.uk

.

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Fortunately, the prospects for developing an effective vaccine to prevent infection with the current H1N1 virus are excellent, and the world’s pharmaceutical companies are working diligently at this task. In contemplating equal access to such a vaccine, it is important to consider three key issues: manufacturing capacity, cost, and delivery.

Only a few countries in the world have plants for manufacturing influenza vaccine, and three companies — GlaxoSmithKline, Sanofi-Aventis, and Novartis — account for most of the world’s manufacturing capacity. The number of doses of vaccine against H1N1 influenza that could be produced with the existing capacity is very large, but the sobering truth is that even if production were switched over completely from seasonal influenza vaccine to pandemic influenza vaccine, there would not be nearly enough for everyone in the world. The size of the gap in potential supply depends greatly on the dose that is required, and it may be possible to reduce the necessary dose by as much as 75% with the use of an adjuvant. The challenging problem is that much, if not most, of the manufacturing capacity is already spoken for through purchasing contracts held by many of the world’s wealthy countries.

The second issue is cost. Despite the enormous technological investment required to create a vaccine, the traditional cost of seasonal influenza vaccines even in wealthy countries is quite low. For the pandemic H1N1 influenza vaccine, the major manufacturers have indicated a willingness to offer tiered pricing, with affordable prices for poor countries. Going even further, Sanofi-Aventis has committed to donating 100 million doses of its vaccine to a stockpile for poor countries, and GlaxoSmithKline has committed to donating 50 million doses. Nevertheless, financial commitments from wealthy countries will be needed to help poorer countries purchase vaccines — cost should not be a barrier to access.

Finally, the scope of access to vaccines will in part be determined by the infrastructure required to deliver them to all citizens in mass campaigns. Ironically, poor countries may have an advantage on this front, since many have recent experience with mass campaigns involving vaccines against polio, measles, and hepatitis B; delivery may therefore be less of a challenge for them, provided that the vaccines reach them in a timely fashion. By contrast, in many wealthier countries, such campaigns have not been undertaken for some time. Getting the vaccine to large numbers of young adults, in particular, may be a formidable task for which preparations must surely be made as soon as possible.

Our limited capacity for producing potentially lifesaving vaccines presents a pressing moral challenge. I believe wholeheartedly that all lives have equal value (this is the basic principle motivating the Bill and Melinda Gates Foundation, where I work), and I believe that every stakeholder has a responsibility to ensure that the pandemic does not take a 1918-like toll on the world. We have therefore worked with partner stakeholders to develop a proposed set of principles to guide the global allocation of pandemic vaccine (see Principles to Guide Global Allocation of Pandemic Vaccine).

Rich countries have a responsibility to stand in line and receive their vaccine allotments alongside poor countries, even if they have paid for their vaccine before others could do so. It would be inexcusable to force poor countries to wait until the rich have been served under their existing contracts with vaccine manufacturers. Moreover, rich countries must also consider how they can provide contributions to offset the cost of vaccines for countries that cannot afford to pay for them. Countries that are home to influenza-vaccine manufacturing plants have a special responsibility to avoid nationalizing those facilities in an effort to reserve their output for their own citizens before others. And all countries must prepare now for the rapid delivery of the vaccines as soon as they become available.

Manufacturers have a responsibility to apply their full capabilities to creating the greatest possible quantity of vaccine doses. Despite contractual obligations to supply many wealthy countries with their vaccines, manufacturers must resist the temptation to commit all their capacity to those who can pay the most. This is not a time to adhere to the “first come, first served” model of business, since we may be facing a health crisis of global proportions in which all people and countries are equally at risk. To ensure fairness, full adherence to a tiered pricing scheme in which the cost to the purchaser is proportionate to its ability to pay is essential. The generous donations made by Sanofi-Aventis and GlaxoSmithKline set an example that all manufacturers should emulate. In return for their responsible actions, it would be reasonable for manufacturers to be indemnified against liability from potential adverse reactions to their vaccines.

Regulatory agencies have an important responsibility in this impending crisis because they stand between the manufacturers of pandemic influenza vaccines and the people who will benefit from them. It is critically important that regulators apply their usual rigorous standards in approving the new vaccines — but also that they do so in a timely fashion. A special task facing them is the rapid review and consideration of the safety and efficacy of adjuvants, whose use could greatly reduce the required dose of vaccine and thereby expand the number of doses that could be manufactured.

The WHO has provided strong leadership as the world has contemplated the prospect of an influenza pandemic. We are counting on the organization to guide us, wisely and fairly, through the complex challenges that lie ahead.

The prospect of a worsening global influenza pandemic is real and will not go away anytime soon. I cannot imagine standing by and watching if, at the time of crisis, the rich live and the poor die. It will take collective commitment and action by all of us to prevent this from happening.

Principles to Guide Global Allocation of Pandemic Vaccine.

1. The global community should take steps to protect all populations, including those without resources to protect themselves.

2. Vaccination should be considered in the context of comprehensive pandemic preparedness and response efforts in all nations.

3. Developed countries and vaccine manufacturers should urgently agree upon a mechanism to ensure access to vaccine by developing countries.

4. Influenza vaccine manufacturers should identify strategies such as tiered pricing and donations to make pandemic vaccine more accessible to developing nations.

5. Pandemic vaccines allocated to developing nations should become available in the same time frame as vaccines for developed nations.

6. The global community should obtain data to help establish a consensus on the safety and efficacy of adjuvants, and efforts should be made to ensure the fullest use of this and other dose-sparing strategies.

7. All countries obtaining pandemic vaccine should ensure that mechanisms are in place to provide the vaccine to their populations, to ensure that this scarce resource is not wasted, and donors should be prepared to provide resources and technical assistance to help countries bolster these mechanisms.

8. The World Health Organization is uniquely positioned to lead the global response to a pandemic virus and should support governments and industry in their efforts to implement these principles.

* From the Pneumonia and Flu Web site of the Bill and Melinda Gates Foundation (www.gatesfoundation.org/topics/Pages/pneumonia-flu.aspx).

Dr. Yamada reports holding equity in GlaxoSmithKline. No other potential conflict of interest relevant to this article was reported.

Source Information

From the Global Health Program, Bill and Melinda Gates Foundation, Seattle.

This article (10.1056/NEJMp0906972) was published on August 12, 2009, at NEJM.org.