MicrobiologyBytes

RNA replicons are derived from either positive- or negative-strand RNA viruses, from which at least one gene encoding an essential structural protein has been deleted. RNA replicons can be regarded as disabled viruses unable to produce infectious progeny. Despite such gene deletions, the viral RNA is replicated and transcribed by the viral RNA polymerase. Any genetic information encoded by the replicon will be amplified many times, resulting in high levels of antigen expression. This property distinguishes RNA replicons from plasmid DNA-based vaccines, which rely on the initial levels of genetic information successfully delivered to the nucleus. The latter together with the characteristics of DNA replication can prove to be a major hurdle for advancing DNA vaccines. Vaccines based on DNA plasmids often contain regulatory sequences and antibiotic resistance genes. The potential integration of such sequences into the host genome by non-homologous recombination may represent an unknown risk. In contrast, replication/transcription of replicon RNA is strictly confined to the cytosol, and does not require any cDNA intermediates, nor is any recombination with or integration into the chromosomal DNA of the host required. Taken together, several safety issues are associated with DNA vaccines, which do not arise with the much more biosafe RNA-based vaccines. Due to autonomous RNA replication, RNA replicons are able to drive high level, cytosolic expression of recombinant antigens stimulating both the humoral and the cellular branch of the immune system. This review provides an update on the available literature covering influenza virus vaccines based on RNA replicons. The pros and cons of this vaccine strategies are discussed.

RNA Replicons – A New Approach for Influenza Virus Immunoprophylaxis. Viruses 2010, 2, 413-434

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Influenza epidemics are the cause of three to five million cases of severe illness every year and approximately 250,000 to 500,000 of these cases are fatal. Epidemics occur during autumn and winter in regions with a temperate climate, while in some tropical countries influenza viruses circulate throughout the year with one or two peaks during the rainy seasons. Mainly the young, elderly and subjects with chronical medical conditions are at risk for developing severe disease after seasonal influenza virus infection. Therefore, the World Health Organization (WHO) recommends annual vaccination of these subjects, which is an effective measure to protect them against influenza and its complications.

The genome of influenza viruses consists of eight gene segments of negative sense RNA and since these viruses lack proofreading activity during their replication, they can accumulate mutations under selective pressure. This way, influenza viruses can escape from recognition by virus-neutralizing antibodies that are induced by previous infections or vaccinations. Indeed, the highest degree of variations is observed in the antigenic sites of the hemagglutinin against which virus neutralizing antibodies are directed. As a result of this antigenic variation, the influenza vaccine that contains components of three currently circulating influenza viruses (A/H1N1, A/H3N2 and B viruses) has to be updated almost every year to match the circulating strains. Since the selection of the vaccine strains and vaccine production has to be carried out before the start of the influenza season, there is some uncertainty in this prediction and mismatches do occur occasionally. In addition to the small gradual antigenic changes of currently circulating influenza virus strains (antigenic drift), occasionally new influenza viruses of novel subtypes are introduced into the human population. The subtypes of these viruses are defined by the envelope glycoproteins of these viruses, the hemagglutinin (HA) and the neuraminidase (NA). Wild aquatic birds are the natural reservoir of all subtypes of influenza from which there is spillover to other (domestic) birds and mammalian species, like pigs, horses and men.

Because antibodies against these viruses are virtually absent in the human population, these viruses may cause pandemic outbreaks of influenza affecting a substantial proportion of the human population. In the last century, three pandemics occurred, which were caused by influenza A viruses of the H1N1, H2N2 and H3N2 subtypes. Recently, influenza A viruses of swine origin have caused the first pandemic of the 21st century. These new pandemic viruses are the result of the exchange of gene segments originating from human, classical swine and avian-like influenza viruses and have emerged and spread worldwide within a few months. As of 30 December 2009 at least 12220 people have been killed due to infection with the influenza A/H1N1(2009) virus. Since not all fatal cases are reported, the real number of fatal cases is most likely much higher.

The current pandemic caused by the new influenza A(H1N1) virus and the current pandemic threat caused by the highly pathogenic avian influenza A viruses of the H5N1 subtype have renewed the interest in the development of vaccines that can induce broad protective immunity. Preferably, vaccines not only provide protection against the homologous strains, but also against heterologous strains, even of another subtype. This paper describes viral targets and the arms of the immune response involved in protection against influenza virus infections such as antibodies directed against the hemagglutinin, neuraminidase and the M2 protein and cellular immune responses directed against the internal viral proteins.

Targets for the Induction of Protective Immunity Against Influenza A Viruses. Viruses 2010, 2(1), 166-188; doi:10.3390/v2010166

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Influenza is circulating in Africa, but virtually no information or attention is evident, says a new essay in this week’s PLoS Medicine. Maria Yazdanbakhsh and Peter Kremsner argue that the lack of adequate surveillance means that the burden of influenza in Africa is incorrectly believed to be negligible. But sporadic reports from various regions in Africa indicate that influenza is circulating and may be regularly causing epidemics. Whereas in temperate areas influenza activity displays a seasonal pattern with marked peaks in the winter, influenza is present all year round throughout the tropics. The authors say that the well-established surveillance network WHO Flu Net in place in Europe and North America, provides continuous data on influenza burden and the spread of viral types and subtypes. Recent threats of pandemic influenza have prompted similar active monitoring in parts of Southeast Asia and Latin America. But the prevalence and incidence of influenza in most tropical countries especially in Africa are largely unknown, say the authors, and improved surveillance is needed. For example, the authors state, the WHO H1N1 swine flu update of May 2009 contained reports of infected patients in many countries, but none in Africa, whereas two reports in October 2009 confirmed swine flu cases from South Africa and Kenya. This indicates that that the virus was circulating in Africa, but because of the lack of a rigorous surveillance system, it was not reported as readily.

Influenza in Africa. 2009 PLoS Med 6(12): e1000182. doi:10.1371/journal.pmed.1000182

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Research published this week in PLoS Medicine presents the most accurate assessment to date of the severity of the swine flu (H1N1) pandemic in the US. Scientists need to measure the severity of swine flu (how often infection with the swine flu virus results in symptoms leading to illness, hospitalization or death) so that appropriate pandemic plans can be put into place. Severity of swine flu has been difficult to measure for two main reasons: first, people with severe influenza are more likely than those with mild cases to seek care, making it difficult to estimate how many total cases have occurred, and second, the sheer number of cases means that recording routine case data can be difficult due to overburdening of public health systems. In this study, researchers from from Milwaukee (where all medically attended cases were recorded, whether hospitalized or not) and New York City (where only hospitalizations, intensive care admission and deaths were recorded, and a telephone survey of flu-like illness was conducted), along with earlier results from studies by the US CDC, used a statistical approach called Bayesian evidence synthesis. This enabled accurate estimations of severity to be made. Their analyses reveal that the autumn-winter pandemic wave of swine flu should have a death toll only slightly higher than, or considerably lower than, that caused by seasonal influenza in an average year, provided swine flu continues to behave as it did during the summer. Seasonal influenza mainly kills elderly adults, but the authors reveal that most deaths from swine flu will occur in non-elderly adults, a shift in age distribution that has been seen in previous pandemics.

The Severity of Pandemic H1N1 Influenza in the United States, from April to July 2009: A bayesian Analysis. PLoS Med 6(12): e1000207 doi:10.1371/journal.pmed.1000207
Accurate measures of the severity of pandemic (H1N1) 2009 influenza (pH1N1) are needed to assess the likely impact of an anticipated resurgence in the autumn in the Northern Hemisphere. Severity has been difficult to measure because jurisdictions with large numbers of deaths and other severe outcomes have had too many cases to assess the total number with confidence. Also, detection of severe cases may be more likely, resulting in overestimation of the severity of an average case. We sought to estimate the probabilities that symptomatic infection would lead to hospitalization, ICU admission, and death by combining data from multiple sources. We used complementary data from two US cities: Milwaukee attempted to identify cases of medically attended infection whether or not they required hospitalization, while New York City focused on the identification of hospitalizations, intensive care admission or mechanical ventilation (hereafter, ICU), and deaths. New York data were used to estimate numerators for ICU and death, and two sources of data – medically attended cases in Milwaukee or self-reported influenza-like illness (ILI) in New York – were used to estimate ratios of symptomatic cases to hospitalizations. Combining these data with estimates of the fraction detected for each level of severity, we estimated the proportion of symptomatic patients who died (symptomatic case-fatality ratio, sCFR), required ICU (sCIR), and required hospitalization (sCHR), overall and by age category. Evidence, prior information, and associated uncertainty were analyzed in a Bayesian evidence synthesis framework. Using medically attended cases and estimates of the proportion of symptomatic cases medically attended, we estimated an sCFR of 0.048% (95% credible interval [CI] 0.026%–0.096%), sCIR of 0.239% (0.134%–0.458%), and sCHR of 1.44% (0.83%–2.64%). Using self-reported ILI, we obtained estimates approximately 7–96lower. sCFR and sCIR appear to be highest in persons aged 18 y and older, and lowest in children aged 5–17 y. sCHR appears to be lowest in persons aged 5–17; our data were too sparse to allow us to determine the group in which it was the highest. These estimates suggest that an autumn–winter pandemic wave of pH1N1 with comparable severity per case could lead to a number of deaths in the range from considerably below that associated with seasonal influenza to slightly higher, but with the greatest impact in children aged 0–4 and adults 18–64. These estimates of impact depend on assumptions about total incidence of infection and would be larger if incidence of symptomatic infection were higher or shifted toward adults, if viral virulence increased, or if suboptimal treatment resulted from stress on the health care system; numbers would decrease if the total proportion of the population symptomatically infected were lower than assumed.

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XVIVO has created a nice animation of influenza virus replication. There are a few small points which are not strictly accurate, but overall, this gives very good impression of the processes which go on which cells are infected with influenza virus.

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CDC have recently published the following fact sheet on Guillain-Barre syndrome (GBS), a rare disorder (wrongly) thought to be frequently associated with influenza infection:

Guillain-Barré syndrome is rare
Guillain-Barré syndrome (GBS) is a rare disorder in which a person’s own immune system damages the nerves, causing muscle weakness and sometimes paralysis. GBS can cause symptoms that last for as little as a few weeks, or go on for several months. Most people recover fully from GBS, but some people have nerve damage that does not go away. In rare cases, people have died of GBS, usually from not being able to breathe due to weakness of their breathing muscles.

GBS may have several causes
While it is not fully known what causes GBS, it is known that about two-thirds of people who get GBS do so several days or weeks after they have been sick with diarrhea or a lung or sinus illness. An infection with the bacterium Campylobacter jejuni, which can cause diarrhea, is one of the most common illnesses linked to GBS. Although rare, people can also get GBS after having the flu or other infections such as Epstein Barr virus. Except for the swine flu vaccine used in 1976, no other flu vaccines have been clearly linked to GBS.

GBS is more common in older adults
Anyone can get GBS, but it is far more common in adults than children. Adults over 50 years of age are 2-3 times more likely to get GBS than younger people. Each year, between 6,000 and 9,100 people in the United States get GBS. This means that about 140 people get GBS every week.

Getting GBS from a vaccination is very rare
In very rare cases, someone may develop GBS in the days or weeks after getting a vaccination. In 1976, there was a small increased chance of GBS after getting a flu (swine flu) vaccination. This means about 1 more case per 100,000 people who got the swine flu vaccine.

Many studies have looked at seasonal flu vaccines and GBS
Since 1976, many studies have been done to see if other flu vaccines may cause GBS. In most studies no link was found between the flu vaccine and GBS. However, two studies did suggest that about 1 more person out of 1 million people vaccinated with seasonal flu vaccine may develop GBS. This continues to be studied.  For the most part, the chance of getting very ill from flu is far higher than the chance of getting GBS after getting the flu vaccine.

CDC has many systems to identify GBS cases
Since GBS is a serious disorder that people get every year, CDC has developed several GBS surveillance systems. These are tracking systems to better see whether some GBS cases are linked to flu vaccinations. During the 2009-2010 flu season, CDC and FDA will be closely looking at reports of serious problems, including GBS, which may be linked to the use of the 2009 H1N1 flu vaccine and to the seasonal flu vaccine. These systems already include some vaccination safety systems, such as the Vaccine Adverse Event Reporting System (VAERS), and new systems, such as the CDC Emerging Infections Program and a partnership with the American Academy of Neurology, which includes doctors who are most likely to see people with GBS. None of these systems existed in 1976. Through these systems, CDC and FDA will be able to find any possible link between GBS and seasonal or 2009 H1N1 flu vaccines early in the vaccination program and take action.

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Influenza viruses contain segmented, negative-strand RNA genomes. Genome segmentation facilitates reassortment between different influenza virus strains infecting the same cell. This phenomenon results in the rapid exchange of RNA segments. In this study, we have developed a method to prevent the free reassortment of influenza A virus RNAs by rewiring their packaging signals. Specific packaging signals for individual influenza virus RNA segments are located in the 5′ and 3′ noncoding regions as well as in the terminal regions of the ORF of an RNA segment. By putting the nonstructural protein (NS)-specific packaging sequences onto the ORF of the hemagglutinin (HA) gene and mutating the packaging regions in the ORF of the HA, we created a chimeric HA segment with the packaging identity of an NS gene. By the same strategy, we made an NS gene with the packaging identity of an HA segment. This rewired virus had the packaging signals for all eight influenza virus RNAs, but it lost the ability to independently reassort its HA or NS gene. A similar approach can be applied to the other influenza A virus segments to diminish their ability to form reassortant viruses.

Rewiring the RNAs of influenza virus to prevent reassortment. PNAS USA September 8 2009 doi:10.1073/pnas.0908897106

For a nice discussion of why this isn’t the answer to influenza pandemics, read What if influenza virus did not reassort? at the Virology Blog.

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• The good news about influenza
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With the fall in H1N1 “swine flu” influenza cases recently, it has become fashionable for the media to run “What was all the fuss about” stories on the same page as “OMG, it’s going to be bad” stories. The problem with influenza is that it is one of the most unpredictable of all viruses, and while an upsurge in the number of cases can be expected as the winter flu season gets going in the northern hemisphere, the real concern is that this new pandemic virus might “turn nasty” in the second wave, just as in 1918 a much more pathogenic variant of that virus followed the relatively benign first wave of cases.

There are two ways in which this could happen. The first is that the present virus acquires spontaneous mutations which make it more pathogenic. The other possibility is that the virus recombines with a highly pathogenic influenza virus though the process known as reassortment – swapping of genes when two different strains infect the same cell. And there’s a good candidate for that out there – the highly pathogenic H5N1 avian influenza virus. Unlike H1N1, H5N1 has a hard time infecting humans, so it’s unlikely that these viruses would meet. But if they did…

The good news comes from Egypt, where H5N1 is relatively common, and a (worrying) case of H5N1/H1N1 co-infection was recently reported. The Ministry of Health has now discounted the rumour of a co-infection with the two viruses. In addition, a University of Maryland/NIH study suggests that co-infections of H1N1 with seasonal flu viruses do not produce chimeric or reassortant viruses. The H1N1 strain seems to outcompete seasonal viruses, possibly demonstrating this pandemic strain is not under biological pressure and is perhaps more efficiently communicable. Certainly, the past pattern seems to suggest that H1N1 pandemic seem to suppress outbreaks of other strains for some time.

A phase I clinical trial conducted by scientists from the University of Leicester tested 100 healthy volunteers with an H1N1 vaccine to see how their immune system responded. Trial leader Dr Iain Stephenson found 80 per cent of the volunteers showed a “strong, potentially protective” response after one dose, with more than 90 per cent showing the same response after two doses. The results suggest that one vaccine dose may be sufficient to protect against A(H1N1) swine flu, rather than two. Larger trials are now under way around the world involving up to more than 6,000 adults and children.

Reasons to be cheeful.

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The genome of the influenza A virus is composed of eight segments of single stranded RNA, which exist as vRNP with nucleocapsid protein (NP) and RNA dependent RNA polymerase. vRNP is surrounded by matrix protein 1 (M1), which is the most abundant protein in influenza virus particles. It is widely accepted that M1 plays significant roles in many aspects of virus life cycle. M1 interacts with RNA and ribonucleoproteins, virus envelope protein, and is involved in transcription inhibition, RNP nuclear import/export, and budding process. M1 expressed on the infected cell surface is responsible for cross reactive recognition by cytotoxic T lymphocytes. Studies suggest that some M1 mutations affect virus particle morphology and are involved in virus-host protein interaction. Several host proteins have been found to associate with M1. These interactions implicate a broad range of biological significances, such as vRNP export and viral morphogenesis. The interactions between M1 and host proteins are critical for viral propagation but it is unknown if M1 protein has any effects on host immune responses.

This paper demonstrates that M1 protein is able to interact with complement C1qA and plays an important inhibitory function in the classical complement pathway. The N terminal domain of M1 protein is required for its binding to the globular region of C1qA. As a consequence, M1 blocks the interaction between C1qA and heat aggregated IgG in vitro and inhibits hemolysis. M1 protein prevents the complement mediated neutralization of influenza virus in vitro. In addition, studies on mice indicate that the administration of M1 can promote a higher virus propagation rate in lung and shortened survival of mice infected with the virus. Taken together, these results strongly suggest that M1 protein plays a critical role in protecting the influenza virus from host innate immune system.

Influenza A virus M1 blocks the classical complement pathway through interacting with C1qA. J Gen Virol. Aug 5 2009

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