Reverse Transcriptase Pcr And Antigen Detection Biology Essay


Detection of AI antigens in clinical specimens by rapid immunochromatographic assays or by direct immunofluorescence are commonly used for diagnosis of human influenza because of their ability for rapid diagnosis. However, the sensitivity of these assay are very low and hence the usefulness of these assays are often limited in detection of infection in patients with avian influenza (Peiris et al., 2004; Yuen et al., 1998). In addition, these rapid antigen detection kits do not help in distinguishing influenza types A and B, and none of the currently available immunochromatographic and immunofluoresent assays distinguish between influenza A subtypes. On the other hand, developments of such H5N1-specific rapid detection assays are ongoing because of their onsite and easy detection capabilities (Xu et al., 2005).

Reverse transcriptase PCR

The Reverse transcriptase (RT-PCR) PCR methods help increase the diagnostic sensitivity and specificity for the detection of viral nucleic acids compared to other culture or antigen detection methods. The RT PCR methods for specific detection of H5N1 viral nucleic acids have proven its applicability as the diagnostic methods of choice during the H5N1 outbreaks in Hong Kong and Southeast Asia (Chotpitayasunondh et al., 2005; Tran et al., 2004; Yuen et al., 1998). The real time RT-PCR methods yield more sensitivity and specificity compared to RT-PCR methods especially when the available viral genomic content is less. In addition, they improve the differentiation within the serotypes of AI, hence real-time PCR technology is a reliable subtype-specific diagnostic method that can be completed within a few hours after specimen collection. The sensitivity of the RT-PCR methods can be greatly improved by designing a stringent serotype specific primers and the risk of false-positive results could be minimized by proper precautions, including physical separation of laboratories for sample preparation and PCR amplification. The use of the uracil-n-glycosylase system also helps to prevent contamination by carryover of amplimers. In addition, the inclusion of an internal control in RT PCR assays is highly desirable to monitor for false-negative results due to inefficient nucleic acid extraction, cDNA synthesis, or amplification.


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The detection of AI subtype specific antibodies is important particularly for epidemiological investigations during outbreaks of avian influenza. Infection neutralizing antibodies were generally detected after 14 days from the onset of symptoms and the antibodies were found to increase till 20 or more days after onset. The Hemagglutination inhibition (HI) assay is the commonly used serology method for detection of antibodies against influenza viruses. However, the sensitivity of the HI assay in detection of antibodies to AI in mammalian species, including humans, seems limited (Beare and Webster, 1991; Hinshaw et al., 1981; Kida et al., 1994) because several studies have failed to demonstrate the presence of HI antibodies In mammals against avian viruses using HI assay, even in cases where infection was confirmed by virus isolation. In another study, it has been demonstrated that HI testing with subunit HA, but not with intact virus, could detect antibodies against an avian H2N2 virus (Lu et al., 1982). However, neutralizing antibodies against the virus could readily be detected with intact virus by microneutralisation assay (MNA). A direct comparison of HI testing with a microneutralization assay in H5N1-infected individuals from the 1997 Hong Kong outbreak indeed showed the MNA to be more sensitive (Rowe et al., 1999) than HI assay. The indirect ELISA using recombinant HA from H5N1/97 showed comparable sensitivity to microneutralization assay, however, the specificity in adult sera was lower which may possibly due to the presence of cross-reactive epitopes common to HAs of almost all AI viruses (Rowe et al., 1999). Based on these observations, it is widely accepted that the neutralization assays are the preferred methods of choice for detection of antibodies against avian viruses in humans. However, the MNA could only be performed at certain laboratories with the stringent biosafety restrictions where handling of live virus is allowed. These assays help to establish the kinetics of the antibody response against H5N1 virus in patients infected during the Hong Kong outbreak are similar to the primary response to human influenza viruses (Katz et al., 1999).

Direct disease control and prophylaxis

The disease control measures should be implemented as soon the outbreak is realized. The initial control measures usually include the proper disposal of infected animals and control of infected material movement. Birds infected with AI excrete large amounts of virus in secretions of animals such as feces and other secretions. This becomes the main source of infectious virus which contaminates water, bird cages vehicle carrying the infected birds, tools, and other fomites in and around the outbreak area and the virus may remain infectious for weeks to months, depending on the humidity and temperature. The virus survives much longer in a humid cold weather than in dry weather where the temperature is above 30°C. Infection in birds caused by HPAI viruses result in systemic replication and the infectious virus often present in their eggs , many tissues and organs. Transmission between birds and bird to human occurs directly or indirectly via contaminated aerosols, feed, water and other materials. However, handling of infected dead poultry or direct exposure to infected live poultry in the week before onset of the illness (Koopmans et al., 2004; Mounts et al., 1999; Tran et al., 2004) is responsible for most, but not all human infections with avian influenza viruses. In a case-control study during H5N1 outbreak indicates that visiting a poultry shop or market selling infected live poultry during the week before the illness as a risk factor, while preparing poultry products or eating were not (Mounts et al., 1999). In cases in which no apparent direct exposure to poultry could be identified, contact with contaminated environment, such as water, has also been suggested (de Jong et al., 2005). Information related to the excretion patterns and periods of potential infectivity in case of human infections are very limited. From the past exposure histories, the incubation time for human H5N1 infections has been estimated to be between 2-10 days, however, it is not clearly understood whether excretion of virus occurs during this time (Tran et al., 2004; Yuen et al., 1998).

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Based on the current understanding on infection control measures during contact with patients with suspected or confirmed infection or with potentially infected birds or environment should prevent closer contact, droplet, and airborne transmission. These measures include the use of high efficiency masks, face shield, gown, or goggles and gloves. Neuraminidase inhibitors are efficient in controlling the infection to some extent because the efficacy of neuraminidase inhibitors against human influenza as seasonal or postexposure prophylaxis is reported to be high (Nicholson et al., 2003). Offering prophylactic treatment to potentially exposed people in the setting of a poultry outbreak of avian influenza, as has been done during H7-outbreaks in the Netherlands and Canada (Koopmans et al., 2004; Tweed et al., 2004), is rational but hardly feasible during the ongoing outbreak in southeast Asia for logistical and financial reasons. In most cases it is advisable that the unprotected healthcare workers and close contacts of infected patients should be given proper postexposure prophylaxis for effective control of the infection. The use of monoclonal antibodies for controlling ongoing infection is already described, but potential use of specific monoclonal antibodies for prophylaxis demand further investigation. Eliminating the source of infection, such as disposal of infected birds and materials which had direct or indirect contact with the infected animals which are suspected to contain the infectious virus particles remains the most effective method for control measure. Elimination of infected birds usually involves culling of all infected folks which has proven successful during avian influenza outbreaks in many parts of the world including Hong Kong, the Netherlands and Canada (Chan, 2002; Koopmans et al., 2004; Tweed et al., 2004). However, considering the different farming practices, the geographic extensiveness and the reported H5N1 infections in migratory birds in Southeast Asia (Chen et al., 2005), it appears that culling of poultry alone will unlikely help contain the outbreak in that region. Based on the current knowledge on disease control measures, it is clearly understood that not a single method alone will help to contain the disease and it is obvious that more than one control measure should be implemented to restrict the disease spread. In addition to proper disposal of virus containing materials, other control measures such as antiviral treatment and vaccination should also be employed for effective control of the disease.


The treatment for AI infection mainly relies on the use of anti-viral drugs.

Antiviral treatment

Currently, there are two classes of drugs with antiviral activity against influenza viruses. One class of drugs is inhibitors of the ion channel activity of the M2 membrane protein which includes amantadine and rimantadine, and the other class is inhibitors of the neuraminidase which includes oseltamivir, and zanamivir. The therapeutic efficacy of amantadine in human influenza is not very well established as only limited number of reliable clinical studies has been performed, however, in individuals, reductions of fever or illness by 1 day have been observed with the use of amantadine (Nicholson et al., 2003). Neurotoxicity is one of the major disadvantages of amantadine. In addition, the development of drug resistance is most common during treatment. The process of development of drug resistance is rapid and resistance is conferred by single nucleotide changes resulting in amino acid substitutions at positions 26, 27, 30, 31, or 34 of the M2 membrane protein. Rimantadine, another drug belongs to the ion channel inhibitors causes less neurological side effects but is not available in several parts of the world. During the 1997 H5N1 outbreak in Hong Kong, several H5N1-infected patients have been treated with amantadine however; the numbers were too small to represent any meaningful conclusions concerning its activity against this virus (Yuen et al., 1998). The genotype Z H5N1 viruses isolated from humans and poultry during an outbreak in Thailand and Viet Nam in 2004 invariably showed an amino acid substitution at position 31 of the M2 protein responsible for conferring amantadine-resistance, indicating that amantadine treatment is not an option even during the ongoing outbreak in southeast Asia (Li et al., 2004; Puthavathana et al., 2005).

The second class of antiviral drugs targeted to neuraminidase inhibitors such as oseltamivir and zanamivir have proven efficacy in treating human influenza when started early during the course of illness, and both drugs are effective particularly as seasonal or post-exposure prohylaxis (Nicholson et al., 2003). The availability of Zanamivir is very poor when administered and therefore it is administered by inhalation. This often limits the use of Zanamivir in the elderly as they may induce bronchospasm. On the other hand, Oseltamivir can be given orally. The development of drug resistance against these drugs during treatment has been reported and is associated with mutations in the active site of neuraminidase or in the haemagglutinin. The latter mutations decrease the affinity of HA for the cellular receptor, thereby obviating the need for neuraminidase to escape the cells.

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The efficacy data available on neuraminidase inhibitors against avian influenza virus are limited. The H5N1 strains which caused the Hong Kong outbreak in 1997 were susceptible to oseltamivir and zanamivir in vitro (Govorkova et al., 2001; Leneva et al., 2000). Oral oseltamivir and topical zanamivir also showed protective and therapeutic activities against Hong Kong H5N1 isolates in murine models (Gubareva et al., 1998; Leneva et al., 2001). These studies also suggest that, higher doses of oseltamivir for longer durations of treatment are necessary to achieve antiviral effects in mice against H5N1 strains causing the Southeast Asian outbreak since 2004. This may be due to higher virulence of H5N1 strains which caused the Southeast Asian outbreak since 2004, when compared to the 1997 Hong Kong H5N1 strain (Yen et al., 2005). Oseltamivir has been used as an option for treatment in several H5N1 infected individuals, but no conclusions can be drawn pertaining to its efficacy. This is however; due to the timing of antiviral treatment may not have been optimal in many cases of avian influenza so far. The antiviral treatment in human influenza was beneficial when the treatment started within 48 h after onset of the illness. But, during the H5N1 outbreak in Viet Nam in 2004, H5N1 infected patients were admitted 5 days or later after onset of symptoms (Tran et al., 2004) and hence proper conclusions could not be made on the efficacy. In addition to Oseltamivir, several H5N1-infected patients have received steroids but the potential benefits of this in clinical studies need further evaluation (Tran et al., 2004). The cytokine dysregulation is considered to be more common in H5N1-infected animals and humans; a beneficial effect of immunomodulating agents could therefore be hypothesized and perhaps requires further investigation.

Monoclonal antibodies

Neutralizing monoclonal antibodies have been shown to be effective in treating established influenza A virus infection in mice with severe combined immunodeficiency (Palladino et al., 1995). However, the reported protocol describes such method in mice but not in human or any other animal. Hence, this strategy needs to be further evaluated before attempting further investigation in human and in poultry against severe illness such as influenza H5N1.


Vaccination has been the most cost effective method of disease prevention against various infectious agents. Vaccination against avian influenza has been extensively studied. The current method of avian influenza vaccine manufacturing involves the cultivation of virus in embryonated eggs followed by inactivation. This method of vaccine manufacturing is well established and hence development of vaccine against almost all the influenza viruses is technically possible. However, production of vaccine against highly pathogenic avian influenza viruses is practically complicated because of the requirement for stringent biosafety containment facilities. In addition, the yield of the viruses after infection in embryonated eggs is very lower which vary further depending on the pathogenicity of individual subtypes of AI. (Stephenson et al., 2004; Wood and Robertson, 2004). Several other approaches have been used in an attempt to overcome these obstacles, including the use of reverse genetics techniques, generation of recombinant haemagglutinin, DNA vaccination and the use of related apathogenic H5 viruses with and without different adjuvants (Nicholson et al., 2003; Stephenson et al., 2004;Webby et al., 2004;Wood and Robertson, 2004). Experimental H5N1 vaccines in which important virulence determinants were altered using plasmid-based reverse genetics, have shown protective efficacy to homologous and heterologous H5 strains in animal models and may prove an attractive approach (Li et al., 1999; Lipatov et al., 2005; Takada et al., 1999). Studies in humans using an H5N3 vaccine developed from a 1997 apathogenic avian virus showed high rates of seroconversions to the vaccine strain and heterologous H5N1 strains after three doses, but only when the vaccine was given with the adjuvans MF59 (Stephenson et al., 2005). In animal models, baculovirus derived recombinant H5 vaccines were immunogenic and protective, but results in humans were disappointing even when using high doses (Crawford et al., 1999; Treanor et al., 2001). H5 DNA vaccines protected mice from infection by homologous, but not by heterologous H5N1 viruses (Epstein et al., 2002; Kodihalli et al., 1999).


An ideal pre-pandemic vaccine

The pre-pandemic vaccine is of utmost important for the effective control of AI infection. An ideal pre-pandemic vaccine should be amenable to rapid, cost-effective, flexible and scalable manufacture, have a method of vaccine administration suitable for mass vaccination, offer protection across antigenically distinct clades, highly immunogenic, eliciting broad immune responses (innate, humoral and cellular) with a low antigen dose delivered in a single shot, be efficacious in individuals of different age groups and health status and have a robust shelf life, preferably at room temperature, thereby avoiding the need for refrigeration (Hoelscher et al., 2008). It is almost impossible to predict the characteristics of the next influenza pandemic strain which necessitates the development and evaluation of efficient pre-pandemic vaccines. The overall preparedness will surely help in controlling the spread of AI infection.

Egg-derived vaccines

Currently, two types of influenza vaccines are manufactured in embryonated chicken eggs which are approved for clinical use. One of them is a live attenuated influenza vaccine (LAIVs) and the other is an inactivated influenza vaccines (whole/ subvirion) cultivated in embryonated eggs followed by inactivation (Ilyinskii et al., 2008). However, in the pandemic circumstance, an egg-derived vaccine has several practical limitations (Gerdil, 2003). The entire process of vaccine manufacture using embryonated eggs takes approximately five to six months from the process of procurement of certified eggs, including the determination of vaccine strains to vaccine release (Gerdil, 2003). Furthermore, seasonal influenza vaccine production are often associated with difficulties in adapting the virus strain for efficient replication in embryonated eggs, while retaining the genetic and antigenic fidelity, which further lengthen the time. During the outbreak, in the event of a pandemic strain arising from an HPAI virus, the potential susceptibility of chickens to HPAI viruses further jeopardizes the availability of eggs for vaccine production. Cell culture based vaccine manufacturing may help overcome the availability of certified eggs for vaccine manufacturing at the time of an ongoing outbreak. Nonetheless, the yields are higher in eggs than in continuous cell lines (Tree et al., 2001). Recent improvements in dose-sparing and production yields have led to the enrichment of the current egg-based vaccine manufacturing capacity by threefold compared to last few years (Roos, 2009).

Cold-adapted, live attenuated vaccines

The cold adopted, FDA approved LAIV against seasonal influenza is an attractive vaccine candidate because of its rapid growth in embryonated eggs. These egg adopted vaccines are generated using an attenuated master donor vaccine strain (A/Ann Arbor/6/1960CaH2N2), which can grow to high titers in eggs with optimum growth temperature at 25- 33°C and reduced replication at 37°C. This master donor virus will be subjected to genetic reassortment with isolates predicted/identified to circulate during a specific influenza outbreak results in virus that express six gene segments derived from the attenuated master donor strain and the remaining two gene segments (haemagglutinin and neuraminidase) derived from the circulating relevant pandemic or epidemic strain. The resulting virus will grow well at 25- 33°C but have reduced ability for systemic replication/disease at 37°C and hence assures maximum level of safety. (US. FDA, 2003). These LAIVs have distinguished advantages over their inactivated counterparts, as they mimic a natural infection resulting in induction of rapid and robust systemic and mucosal immune responses (Tosh et al., 2008).

The LAIVs have also been demonstrated to be efficacious in chronically ill subjects and some high-risk populations suffering from chronic obstructive pulmonary disease (COPD) (Gorse et al., 1988; Gorse et al., 1991; Treanor et al., 1999). The efficacy of LAIVs has already been evaluated in children with and without recurrent respiratory tract infection and asthma (Belshe et al., 2007; Ashkenazi et al., 2006; Fleming et al., 2006). A comparative study in infants and young children between trivalent LAIV consisting of three influenza strains (two different type A and one type B influenza strains) and an inactivated trivalent influenza vaccine consisting recombinant 2004:2005 influenza strains demonstrated comparable safety, but better efficacy for the LAIV (Belshe et al., 2007). Furthermore, the LAIVs also reported to confer some degree of cross-protection (Belshe et al., 2000; Gerdil, 2003) in children. Another study in children also demonstrated that the intranasal administration of LAIVs for vaccinating children. These makes this approach ideal for vaccinating children and preclude the need for trained personnel for vaccine administration, thus making it feasible to rapid mass vaccination in a pandemic situation. In addition, a higher amount of LAIVs per egg can be produced than for the trivalent inactivated influenza vaccine. This will indeed help enhancing the vaccine coverage with the existing manufacturing capacity and offering a potential advantage in terms of pandemic preparedness.

Despite these potential advantages, several concerns relating to adverse reactions to the vaccine, reversion to wild type, virus shedding and reduced efficacy have arisen over the years which have led to limited acceptability of LAIVs. On the other hand, many of such concerns were subjected to argument while highlighting the importance over the disadvantages of LAIVs (Tosh et al., 2008). With recent gene manipulation techniques, developing vaccine candidate strains without recourse to reassortant methods are possible. For example, modifying the polybasic cleavage site on the haemagglutinin gene which is associated with high pathogenicity and virus spread in birds can be derived from HPAI H5N1 viruses (Gabriel et al., 2008; Govorkova et al., 2005; Nicolson et al., 2005; Horimoto and Kawaoka, 1994; Senne et al., 1996). Development of many such H5N1 LAIVs has shown promising outcome in preclinical studies (Steel et al., 2009; Suguitan et al., 2006) particularly for H5N1 LAIVs generated by reverse genetics. A H5N1 LAIVs developed by this method consisted the gene segments derived from the donor strain, A/Ann Arbor/6/1960 ca (H2N2) but haemagglutinin and neuraminidase derived from H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003 and 2004, which demonstrated complete protection in ferrets and mice against both homologous and antigenically heterologous H5N1 virus challenge (Suguitan et al., 2006). Similarly, a single dose of a LPAI H7N3 cold adopted live attenuated virus generated by reverse genetics was completely protective in ferrets and mice against both homologous and antigenically heterologous H7 virus challenge (Joseph et al., 2008). In addition to modification of the polybasic cleavage site within haemagglutinin, manipulation of other pathogenic determinants of these HPAI viruses have also been used to develop LAIVs (Steel et al., 2009). Specifically, an H5N1 LAIV based on A/ Vietnam/1203/2004 was attenuated through modification of the haemagglutinin cleavage site, a truncation of the C-terminus of nonstructural (NS1) protein, and an amino acid substitution in the PB2 polymerase. This vaccine provided complete protection in mice and chickens following challenge with homologous H5N1 virus (Steel et al., 2009). Unfortunately, the success of the seasonal trivalent LAIV has not been replicated in the pandemic vaccine candidate H5N1 LAIVs. To meet the requirement of the European Committee for Proprietary Medicinal Products (CHMP) for immunogenicity, for adults the seroprotection rate must exceed 70% and the seroconversion rate must be at least 40% and for elderly subjects, the respective limits are 60% and 30%. In a clinical trial, only 11% of the study subjects aged 18-49 years seroconverted following two doses of A/ Vietnam/ 1203/2004 - A/Ann Arbor/6/1960 ca vaccine (WHO, 2009). A similar vaccine based on A/chicken/ British Columbia/CN-6/2004 H7N3 - A/Ann Arbor/6/1960 ca resulted in a 1.6-fold increase in HI titres in 62% of study participants (WHO, 2009). In addition, a high-growth reassortant Len17/H5 vaccine that contained the haemagglutinin gene from the nonpathogenic virus A/Duck/Potsdam/14026/1986 (H5N2) and other genes from the ca attenuated A/Leningrad/134/17/1957 (H2N2) strain (Ref. 69) resulted in 6% and 47% of subjects with fourfold or higher HI titres after one or two doses, respectively (WHO, 2009). However, recently a candidate H9N2 LAIV (A/chicken/ HongKong/G9/1997 - A/AnnArbor/ 6/1960 ca) looked promising in a Phase I clinical trial wherein the vaccine generated a fourfold increase in HI titers in 92% of sero-negative adult subjects under the age of 40 (WHO, 2009).

Whole-virus inactivated vaccines

The whole-virus inactivated vaccines have been prepared similar to that described for LAIV using genetic reassortment with the HA and NA genes derived from the appropriate circulating strain, but the only major difference being that the genes other than HA and NA are from the high-yielding donor strain A/Puerto Rico/8/1934. Because the master donor virus for this type has a optimum growth temperature of 37°C, this virus grows faster in embryonated eggs than LAIVs which after propagation will be subjected to formalin inactivation (Ref. 73). Vaccine preparations consisting chemically disrupted influenza virus or subunit preparations containing antigenic proteins purified after chemical solubilisation of influenza virus have been preferred especially in children. This preference is because of the hypo-reactogenicity of such split vaccine preparation over whole-virus inactivated vaccines (Ref 74,75,76). However, an egg derived, inactivated H9N2 whole virus vaccine was shown to produce better responses in a naïve population with a single dose than its subunit counterpart (Ref. 77). Furthermore, use of whole-virus vaccines may also avoid the antigen losses during the disruption process of an inactivated split-virion vaccine (Ref. 73). An alum-adjuvanted inactivated whole-virus H5N1 vaccine was well tolerated and resulted in 78% seropositivity (proportion of individuals achieving HI titres of ≥40) by day 42 with a two dose regimen of 10 mg per dose (Ref. 73) (Table 1). Recently, the same vaccine was shown to be broadly cross-protective after two inoculations with 10 mg in 98% and 87% of subjects against heterologous A/Indonesia/5/2005 and A/Anhui/ 1/2005 clade 2 strains, respectively (Ref. 78). A clade 1 A/Vietnam/1194/2004 NIBRG-14 H5N1 whole-virus prototype pandemic influenza vaccine, derived by reverse genetics and formulated with an aluminum phosphate adjuvant, was tested in 88 subjects [44 adults (19-60 years) and 44 elderly (60-83 years)], who received a single dose (6 mg) of the vaccine. Vaccine recipients demonstrated seroconversion in 90% and 68% of the adult and elderly subjects, respectively, against the homologous virus and also showed significant crossreactive immune responses against the different clade 2 H5N1 viruses in both age groups. Interestingly, higher titres of virus-neutralising antibodies and crossreactive antibodies were detected in the elderly, and a surprising 56% of the elderly subjects also seroconverted against an H1N1 strain, compared with only 18% of adults, suggesting the probable importance of regular vaccination with seasonal influenza vaccines in developing crossreactive immunity against pandemic viruses (Ref. 79). This vaccine was shown to be safe and immunogenic in the first clinical trial in children, and of the 12 healthy children (9-17 years) who received a single dose of 6 mg of vaccine, 75% seroconverted 21 days after vaccination (Ref. 80). Owing to the inherent low immunogenicity of H5 haemagglutinin in humans, novel adjuvanted formulations of inactivated whole-virus vaccine will not only enhance the immunogenicity but also lead to dose-sparing. However, a potential safety concern regarding the use of an adjuvanted inactivated influenza vaccine became apparent when an intranasal vaccine formulation introduced in Switzerland was strongly associated with Bell palsy (Ref. 81), suggesting the need for a large-scale safety trial for the licensure and commercial release of a new vaccine formulation.

Inactivated split-virus vaccines

One of the first studies that evaluated the safety and immunogenicity of an inactivated, unadjuvanted subvirion H5N1 vaccine (A/Vietnam/ 1203/2004 - A/Puerto Rico/8/1934) in a clinical setting demonstrated that high doses (two 90 mg doses) were required for developing protective antibody titres (Ref. 82). Nonetheless, this vaccine was subsequently approved by the FDA and has the distinction of being the first avian influenza vaccine available in the USA (Ref. 83). In a sequel to this study, a third dose of the vaccine, administered six months after the second dose, resulted in protective antibody titres in 78% and 67% of the subjects who initially received 90 or 45 mg doses, respectively. The neutralising antibody levels remained significantly higher even five months after the third dose, compared with that observed after the second dose (Ref. 84). It should be noted, however, that increasing the amount of antigen and the number of doses is not a viable strategy for pandemic preparedness both in terms of time and economics, and therefore dose-sparing using novel adjuvant strategies will be required (Refs 85,86,87,88,89). Alum is the only approved adjuvant for use in the USA in human vaccine formulations, but alum-adjuvanted inactivated influenza vaccines have demonstrated variable results, with effects ranging from moderate to none (Refs 90,91,92). An alum-adjuvanted inactivated splitvirion vaccine (A/Vietnam/1194/2004) elicited 67% seroconversion at the highest dose (30 mg) tested (Ref. 90). Other clinical trials evaluated the immunogenicity of a nonpathogenic H5N3 virus (A/Duck/Singapore/1997) adjuvanted with the squalene-based oil-in-water emulsion MF59 (Refs 87,93,94). This study demonstrated significantly higher seroconversion at a dose of 7.5 mg in the adjuvanted vaccine group compared with the nonadjuvanted vaccine group (Ref. 87). Recently, a promising dose-sparing study using an egg-derived, inactivated split vaccine (A/Vietnam/1194/2004 - A/Puerto Rico/8/1934) along with a proprietary oil-inwater emulsion-based adjuvant elicited 86% seroconversion at the lowest dose of 3.8 mg given twice (Ref. 85). Interestingly, 77% of the study subjects seroconverted for neutralising antibody titres against an antigenically dissimilar clade 2 virus, highlighting the potential for crossclade seroprotective immune responses at a low antigen dose (Ref. 85). The crossclade protective efficacy of this vaccine was amply demonstrated in a ferret model (Ref. 95). The safety and reactogenicity profile of a 15 mg haemagglutinin dose of the same proprietary oil-in-water emulsion-adjuvanted split virion (A/Vietnam/1194/2004 NIBRG-14 H5N1) vaccine preparation was evaluated in a multicentre, randomised, Phase III clinical trial in healthy adults and was considered clinically acceptable in the context of its use against pandemic influenza (Ref. 96). Another clinical trial evaluating an even lower dose (1.9 mg) of an influenza A/ Vietnam/1194/2004 NIBRG-14 (H5N1) vaccine given along with a 5% squalene-in-water emulsion-based adjuvant showed seroconversion in 72% of the study subjects (Ref. 86).

A clinical trial involving 486 healthy adults (aged 18-60 years) and elderly individuals (60 years and over) with an MF59-adjuvanted clade 1(A/Vietnam/1194/2004) inactivated subunit H5N1 vaccine demonstrated comparable seroprotection in both adult and elderly populations after two doses of 7.5 or 15 mg of vaccine, ranging between 72% and 87% (Ref. 97). There was a significant reduction in seroconversion after six months in both the adults and elderly.

However, the group that received a third booster dose (7.5 or 15 mg) showed seroprotection of 90% and 84% or better in the adult and elderly groups, respectively. This vaccine formulation also generated crossneutralising antibodies to a clade 2 H5N1 virus, suggesting that it can be used in a prepandemic scenario and, more importantly, for all age groups (Ref. 97). Given the potentially serious course of influenza in children, the efficacy of the H5N1 influenza vaccine has also been evaluated in infants and children (Refs 98,99). Two 30 mg doses of an aluminiumadjuvanted, inactivated, split-virus, clade 1 (A/Vietnam/1194/2004 NIBRG-14) H5N1 vaccine given in infants and children (N=150) aged six months to nine years was well tolerated and elicited a strong neutralising antibody response in 99% of subjects, with persistence of neutralising antibody response up to six months after vaccination in 85% of the children. Furthermore, 80% of the subjects showed crossreactivity to a clade 2.1 variant strain (A/Indonesia/5/2005 CDCRG2) (Ref. 99). Similarly, a Phase II clinical trial involving children (N=180) aged six months to 17 years given two 30 mg doses of an aluminiumhydroxideadjuvanted H5N1 influenza vaccine (A/Vietnam/1194/2004 NIBRG-14) administered 21 days apart showed seroresponses in 79% of the subjects (Ref. 98).

The concept of proactive prepandemic priming that may induce long-lasting immune memory and allow a single booster vaccine to induce protection has gained momentum as a potential strategy for pandemic preparedness (Ref. 100). Two 7.5 mg booster doses of an inactivated MF59-adjuvanted A/Vietnam/1194/ 2004 NIBRG-M vaccine were administered in subjects who had previously (at least six years earlier) been primed with two doses of either MF59- adjuvanted or nonadjuvanted A/duck/ Singapore/1997 (H5N3) vaccine containing 7.5- 30 mg of haemagglutinin. These booster doses induced a significantly better vaccine response in subjects primed with MF59-adjuvanted vaccine in terms of early induction and persistence of crossreacting antibody responses for six months than in unprimed subjects or in those primed with an unadjuvanted vaccine. Seven days after administration of the booster, 90% of MF59- primed subjects exhibited high titres of neutralising antibodies towards diverse H5N1 viruses, as well as to the original H5 priming strains, suggesting H5 priming with MF59-adjuvanted vaccine may result in dose-sparing by limiting the amount and number of doses required to achieve seroprotective titres from a distinct H5 strain. This might be due to the induction of an immune memory B cell pool that probably expands more rapidly and efficiently with booster immunisation (Refs 101,102). These recent findings have highlighted the importance of novel adjuvant formulations and prime- boost strategies in influenza pandemic preparedness.

Egg-independent vaccine strategies

Cell-derived whole-virus inactivated vaccines

As a part of pandemic preparedness, the US Department of Health and Human Services has offered substantial monetary incentives to the vaccine industry to develop cell-based influenza vaccine infrastructure in the USA (Refs 103,104). Major vaccine manufacturers are at various stages of developing cell-derived vaccines for seasonal and pandemic influenza (Refs 103,104), and a cell-derived seasonal influenza vaccine is already on the market in Europe (Ref. 105). Cell-derived vaccines assume much significance with respect to pandemic preparedness since their production is not exposed to anticipated vulnerabilities in the supply of embryonated eggs in a prepandemic/ pandemic scenario. Additionally, cell-based vaccine manufacturing in closed scalable bioreactors reduces the risk of exogenous contamination. Following the identification of the pandemic strain, it has a shorter lead manufacturing time of one to three months compared with approximately five to six months for the egg-based approach (Ref. 106). The cell-based production of influenza vaccines minimises the selection of any virus variants with reduced immunogenicity and altered antigenicity, which might sometimes result during the adaptation of virus strains to eggs in an egg-based vaccine manufacturing process (Refs 107,108,109). However, some of the limitations of the cell-based vaccine approach are the lack of standardised reagents and the potential safety concern regarding the introduction of adventitious agents during the cell-based vaccine manufacturing process (Ref. 110). To date, three cell lines - African green monkey kidney (Vero), Madin-Darby canine kidney (MDCK) and human PER.C6 - are approved for influenza vaccine production, and influenza vaccines produced using these cell lines have been tested in several preclinical trials (Refs 111,112,113). Vero-cell-derived whole-virus inactivated vaccine based on clade 1 (A/Vietnam/ 1203/2004) and clade 2 (A/Indonesia/ 5/2005) viruses was shown to induce crossneutralising antibodies and highly crossreactive T cells, and protected mice from challenge with antigenically divergent H5N1 viruses (Ref. 112). In a dose-escalation Phase I/ II clinical trial, two 7.5 mg doses of the same Vero-cell-derived clade 1 (A/Vietnam/1203/ 2004) inactivated whole-virus vaccine resulted in virus neutralisation titres of ≥20 in 76% of the study subjects (Ref. 114). Importantly, the vaccine also induced crossneutralising antibody titres (≥20) in 76% of subjects against clade 0 (A/Hong Kong/156/1997) and in 45% against clade 2 (A/Indonesia/ 05/2005) viruses (Ref. 114). Advanced Phase III clinical trials are currently under way to test this vaccine in adults, the elderly and specified risk groups, such as human immunodeficiency virus (HIV)infected individuals and chronically ill patients (Refs 115,116). However, the use of such a cell-derived whole-virus H5N1 vaccine with an intact haemagglutinin polybasic cleavage site represents a potential public health hazard.

Recombinant-protein-based vaccines-Generation of antigenic protein by recombinant baculoviruses in insect cells is a convenient and fast method to generate large amounts of haemagglutinin protein for influenza vaccines (Ref. 117) (Fig. 2). Baculovirusmediated protein expression is extremely efficient under a strong polyhedrin promoter, and the expressed protein undergoes post-translational modifications similar to those occurring in mammalian cells (Refs 118,119). Several clinical trials have demonstrated the safety and immunogenicity of baculovirus-expressed recombinant haemagglutinin (Refs 120,121,122, 123,124,125). Two 90 mg doses of a purified recombinant haemagglutinin from a clade 0 H5N1 virus (A/Hong Kong/156/ 1997) generated potentially protective neutralising antibody titres (1:80) in 52% of the naive subjects (Refs 125,126). In a follow-up study eight years later in the same subjects, 68% of the subjects (now clade-0-primed), induced potentially protective titres (≥1:40) when immunised with a single 90 mg dose of an egg-derived inactivated subvirion clade 1 vaccine (A/Vietnam/1203/2004 CDCRG1) as compared with only 45% in nonprime subjects receiving two 90 mg doses of the same vaccine (Ref. 126). This suggests that prepandemic priming may reduce the global vaccine burden in a pandemic scenario. Use of better adjuvants and conserved internal protein(s) in the recombinant haemagglutinin vaccine formulations may further broaden the vaccine efficacy.

Virus-like particle (VLP)-based vaccines-VLP vaccines are highly organised particles derived from the self-assembly of viral structural proteins that lack viral nucleic acid and hence are completely noninfectious. Thus, they mimic live viruses and can induce robust humoral and cellular immune responses (Ref. 127), and can be manufactured in bulk using a baculovirus expression system and insect cells (Refs 128,129) (Fig. 2). Recently, a VLP-based human papilloma virus (HPV) vaccine was launched for commercial use, emphasising the safety and immunogenicity of this approach (Refs 130,131). Furthermore, the safety and protective efficacy of this approach for the development of pandemic influenza vaccines has been evaluated in several preclinical trials (Refs 132,133,134,135). An H5N1 A/Indonesia/5/2005 VLP vaccine evaluated for its immunogenicity and safety in a Phase I/IIa clinical trial was well tolerated and immunogenic in healthy adult subjects (Ref. 136). The vaccine dose (15, 45 or 90 mg) showed a dose-dependent increase in HI titres, and the highest dose of 90 mg resulted in seroconversion (HI titres ≥ 1:40) in 63% of the study subjects (Ref. 136). The protection coverage offered by VLP influenza vaccines can be further broadened by developing multivalent VLP vaccines incorporating haemagglutinin from different influenza virus subtypes (Ref. 137).

DNA vaccines-DNA vaccine technology enables easy manipulation to incorporate single or multiple genes of interest in a plasmid (Fig. 2). Furthermore, molecules that can modulate or stimulate the immune response can also be incorporated in the same plasmid to further boost the vaccine efficacy. DNA vaccines are capable of inducing both humoral and cellular immune responses with either a predominant T helper 1 (Th1)-or Th2-type response, depending on the route of DNA delivery (specifically, intramuscular injection versus gene gun delivery to the skin, respectively) (Refs 138,139). While demonstrating protective efficacy in a mouse model (Ref. 140) and a good safety profile (Ref. 141), DNA vaccines have suffered from inefficient gene delivery methods resulting in suboptimal immune responses (Ref. 142). Currently, electroporation or bombardment with particles coated with DNA into the skin are the preferred methods for the delivery of DNA vaccines (Refs 143,144). Nonetheless, development of efficient delivery systems that may result in better antigen expression and subsequent immune responses are needed (Ref. 145). DNA vaccines aiming to induce crossprotective immune responses by incorporating multiple haemagglutinin genes delivered simultaneously (Ref. 146), using a consensus haemagglutinin sequence (Ref. 147), or expressing highly conserved influenza virus proteins (such as nucleoprotein or M2) are currently being evaluated in preclinical trials (Refs 148,149,150, 151). A trivalent Vaxfectin-formulated DNA vaccine (Refs 152,153) encoding H5 haemagglutinin (A/Vietnam/1203/2004), nucleoprotein and M2 consensus sequences administered intramuscularly or with a Biojector 2000 injection system was shown to be safe and efficacious in a Phase I clinical trial (Ref. 154). The vaccine induced protective antibody titres in 67% of the study subjects receiving two doses of 0.5 or 1.0 mg of vaccine (Ref. 154). The protective efficacy of a three-dose regimen of a DNA vaccine encoding H5 haemagglutinin (A/Indonesia/5/2005) following intramuscular inoculation using a Biojector is currently being evaluated (Ref. 155). The safety, rapid scalability and well-defined manufacturability of DNA vaccines make this approach suitable for developing vaccines against emerging infectious diseases like influenza.

Viral-vector-based vaccines-Viral vectors act as delivery vehicles that carry the gene(s) of interest into the host cell for efficient expression (Fig. 2). They serve as a live vaccine and induce robust immune responses without the associated risk of pathogenicity/infection. Viral vectors, by virtue of innate immune activators, also exert an adjuvant effect, thereby boosting the vaccine efficacy. Furthermore, the ease of incorporating multiple genes/adjuvants in a single vector may offer flexibility in vaccine design. Viral vectors can be grown to high titres in qualified cell lines in large quantities. Vectors based on a range of viruses are currently under consideration for influenza vaccines, with adenovirus-based vectors receiving particular attention.

Adenoviruses possess several attributes that make them suitable candidates for vaccine vectors. They are nonpathogenic viruses capable of infecting both dividing and nondividing cells, facilitating high levels of transgene expression without integration into the host genome, and more importantly can be grown to high titres in qualified cell lines (Ref. 156). Adenoviruses exert an adjuvant-like effect by stimulating the innate immune system through both Toll-likereceptor- dependent and -independent pathways (Ref. 157). The effectiveness of an adenovirus (Ad)-vectored vaccines against many infectious diseases, including measles, severe acute respiratory syndrome (SARS), HIV, hepatitis B and Ebola, has been evaluated in human and animal models (Refs 158,159,160,161,162). The viability of a human Ad (HAd)-vectored vaccine for H5N1 pandemic influenza vaccine (HAd-H5HA) in mouse and chicken models has been demonstrated (Refs 163,164,165). Two doses of 1 - 108 plaque-forming unit (PFU) of HAd-H5HA expressing the haemagglutinin gene of A/Hong Kong/156/1997 generated significantly high levels of virus-neutralising antibody titres, and elicited a significantly increased haemagglutinin-epitope-specific CD8+ T cell population secreting interferon γ, in a mouse model (Ref. 164). The vaccine protected mice against lethal challenge with homologous as well as antigenically distinct H5N1 influenza viruses (Ref. 164). The immune responses humoral/cell-mediated) and the protective efficacy of HAd-H5HA vaccine persisted for at least 12 months after immunisation in mice (Ref. 166). Inclusion of haemagglutinin from clade 1 and clade 2 as well as the conserved nucleoprotein in the vaccine formulation further broadened the protective efficacy of HAdbased H5N1 vaccines, thereby offering complete protection in mice against challenge with either clade 1 or clade 2 virus (Ref. 167). Ad-vector-based influenza vaccines have been shown to be safe and immunogenic in humans (Refs 45,168), and intranasal administration of a two-dose regimen of 5 - 108 virus particles expressing haemagglutinin from A/Puerto Rico/8/1934 resulted in a fourfold increase in HI titres in 83% of the subjects in a clinical trial (Ref. 45). Several preclinical studies have suggested HAd-vector immunity (the presence of pre-existing HAd-specific neutralizing antibodies) might inhibit generation of immune responses against the transgene product (Refs 156,169), but a clinical trial specifically demonstrated that increasing the vaccine dose of an HAd-based HIV vaccine overcame the HAd-vector immunity (Ref. 170). Nonetheless, the use of nonhuman Ad-vector-based vaccines, either alone or as the boost component in a DNAprime and Ad-vector-boost immunisation strategy using a combination of DNA and Ad-vector antigen delivery systems (Refs 156,171,172,173,174), is among several alternative approaches that are being explored to circumvent the limitations of HAd-vector immunity. A bovine-origin Ad vector (bovine Ad subtype 3 or BAd)-based H5N1 vaccine (BAd-H5N1) has been shown to circumvent high levels of pre-existing HAd-vector immunity in a mouse model (Ref. 173), and this vaccine completely protected mice against homologous H5N1 virus challenge (Ref. 173). In addition, a prime-boost strategy using HAd and BAd vectors generated better immune

responses compared with the responses generated with either vector alone, suggesting the importance of two vector systems in improving vaccine-induced immune responses (Ref. 173). Other viral vectors of potential interest for influenza vaccines include those based on alphavirus, Newcastle disease virus (NDV), vesicular stomatitis virus (VSV) and pox virus. Vectors based on alphaviruses (positive-strand RNA viruses) have primarily been developed using Venezuelan equine encephalitis, Sindbis or Semliki Forest viruses (Ref. 175), and have shown protection in numerous models for infectious diseases, including influenza, HPV and Ebola (Refs 176,177,178,139). Alphavirus replicon particles expressing the haemagglutinin gene from an H5N1 isolate (A/Hong Kong/156/1997) were shown to be protective in chickens (Ref. 177). Another alphavirus, expressing haemagglutinin from A/ Wyoming/03/2003 (H3N2), was immunogenic in preclinical studies in mice, rabbits and macaques (Ref. 179). A Phase I clinical trial using the same H3N2 vaccine induced protective HI titres in 77% and 80% of subjects receiving a single dose of low-and high-concentration vaccine, respectively (Ref. 180). A second immunisation in these individuals enhanced seroprotective responses to 86% for both dosage levels and extended the duration of T cell responses as compared with the single immunisation (Ref. 180). NDV vectors have demonstrated immunogenicity and protective efficacy against HPAI in several preclinical studies in mouse and chicken models (Refs 181,182). Reverse genetics technology has helped in developing NDV as a safe and efficacious vaccine vector (Ref. 183), and both the natural mucosal route of infection by NDV and the high degree of attenuation of this virus in primates make it suitable as a vaccine vector for humans for the control of respiratory virus infections such as influenza (Ref. 184). A live attenuated NDV-based vaccine expressing haemagglutinin (NDV-HA) of highly pathogenic H5N1 virus (A/Vietnam/ 1203/2004) was highly attenuated in nonhuman primates, and a single inoculation by both the intranasal and intratracheal routes with 107 PFU of NDV-HA per site induced substantial serum IgG and mucosal IgA responses (Ref. 185). VSV vectors have been developed as potential therapeutic and vaccine vectors for various diseases (Refs 186,187,188,189,190,191,192). A recombinant VSV expressing haemagglutinin of an H5N1 virus induced robust neutralising antibody titres against the homologous and more recent antigenically distinct H5N1 viruses in mice (Ref. 193). A single dose of the vaccine offered durable protection for up to seven months against lethal challenge with the homologous H5N1 virus (Ref. 193). The VSV vector expressing haemagglutinin of HPAI A/ FPV/Rostock/ 1934 (H7N1), in place of the VSV G gene, protected chickens against challenge with the homologous H7N1 virus but not against the H5N2 virus (Ref. 194). Low seroprevalence of VSV in humans makes it a suitable vector for human applications, but more preclinical studies are required to prove its usefulness for the design of a pandemic influenza vaccine.

Poxvirus vectors can accommodate large or multiple gene inserts of approximately 25 kb in size (Refs 195,196). Modified vaccinia virus Ankara (MVA) recombinants expressing influenza virus antigens have been evaluated in several preclinical trials (Refs 197,198,199). A vaccinia-virus-based multivalent H5N1 influenza vaccine expressing haemagglutinin, neuraminidase and nucleoprotein from A/Vietnam/1203/2004 and M1 and M2 from A/CK/ Indonesia/PA/2003, and adjuvanted with interleukin 15, elicited protective neutralizing antibody titres (1:80) against both clade 1 and clade 2.2 viruses in mice (Ref. 200). Appreciable amounts of influenza-specific antibody responses were detectable in mice for up to 14 months after vaccination (Ref. 200). Similarly, a single dose of a nonadjuvanted replication-defective vaccinia H5N1 vaccine based on A/ Vietnam/1203/2004 induced substantial cell-mediated immune responses and provided crossclade protection in mice (Ref. 201).

Pichia pastoris as an expression platform

P. pastoris is a single-celled microorganism that is easy to manipulate and culture. However, it is also a eukaryote and capable of many of the posttranslational modifications performed by higher eukaryotic cells, such as proteolytic processing, folding, disulfide bond formation, and glycosylation. Thus, many proteins that end up as inactive inclusion bodies in bacterial systems are produced as biologically active molecules in P. pastoris. The P. pastoris system is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from higher eukaryotes, such as insect and mammalian tissue culture cell systems, and usually gives higher expression levels.

A second role played by P. pastoris in research is not directly related to its use as a protein expression system. P. pastoris serves as a useful model system to investigate certain areas of modern cell biology, including the molecular mechanisms involved in:

• the import and assembly of peroxisomes;

• the selective autophagic degradation of peroxisomes; and

• the organization and function of the secretory pathway in eukaryotes.

The methanol metabolic pathway appears to be the same in all yeasts and involves a unique set of pathway enzymes (21). The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde, generating hydrogen peroxide in the process, by the enzyme alcohol oxidase (AOX). To avoid hydrogen peroxide toxicity, this first step in methanol metabolism takes place within a specialized organelle, called the peroxisome, which sequesters toxic hydrogen peroxide away from the rest of the cell. AOX is a homo-octomer with each subunit containing one noncovalently bound FAD (flavin adenine dinucleotide) cofactor. Alcohol oxidase has a poor affinity for O2, and methylotrophic yeasts appear to compensate for this deficiency by synthesizing large amounts of the enzyme.

There are two genes in P. pastoris that code for AOX-AOX1 and AOX2-but the AOX1 gene is responsible for the vast majority of alcohol oxidase

activity in the cell (18). Expression of the AOX1 gene is tightly regulated and induced by methanol to high levels. In methanol-grown shake-flask cultures, this level is typically approx 5% of total soluble protein but can be ≥30% in cells fed methanol at growth limiting rates in fermentor cultures (22). Expression of the AOX1 gene is controlled at the level of transcription (12,16,18). In methanol-grown cells, approx 5% of polyA+ RNA is from the AOX1 gene, whereas, in cells grown on other carbon sources, the AOX1 message is undetectable. The regulation of the AOX1 gene is similar to the regulation of the GAL1 gene of S. cerevisiae, in that control appears to involve two mechanisms: a repression/derepression mechanism plus an induction mechanism. However, unlike GAL1 regulation, derepressing conditions (e.g., the absence of a repressing carbon source such as glucose in the medium) do not result in substantial transcription of the AOX1 gene. The presence of methanol appears to be essential to induce high levels of transcription (16).

P. pastoris has the potential to perform many of the posttranslational modifications typically associated with higher eukaryotes. These include processing of signal sequences (both pre- and prepro-type), folding, disulfide bridge formation (10,11,36), and O- and N-linked glycosylation.