Robert Lambkin

A plant-produced influenza subunit vaccine protects ferrets against virus challenge

Background  Influenza A viruses are of major concern for public health, causing worldwide epidemics associated with high morbidity and mortality. Vaccines are critical for protection against influenza, but given the recent emergence of new strains with pandemic potential, and some limitations of the current production systems, there is a need for new approaches for vaccine development.

Human Genetics and Responses to Influenza Vaccination

Influenza A and B viruses are negative-strand RNA viruses that cause regular outbreaks of respiratory disease and substantially impact on morbidity and mortality. Our primary defense against the influenza virus infection is provided by neutralizing antibodies that inhibit the function of the virus surface coat proteins hemagglutinin and neuraminidase. Production of these antibodies by B lymphocytes requires help from CD4+ T cells. The most commonly used vaccines against the influenza virus comprise purified preparations of hemagglutinin and neuraminidase, and are designed to induce a protective neutralizing antibody response. Because of regular antigenic change in these proteins (drift and shift mutation), the vaccines have to be administered on an annual basis. Current defense strategies center on prophylactic vaccination of those individuals who are considered to be most at risk from the serious complications of infection (principally individuals aged >65 years and those with chronic respiratory, cardiac, or metabolic disease).The clinical effectiveness of influenza virus vaccination is dependent on several vaccine-related factors, including the quantity of hemagglutinin within the vaccine, the number of doses administered, and the route of immunization. In addition, the immunocompetence of the recipient, their previous exposure to influenza virus and influenza virus vaccines, and the closeness of the match between the vaccine and circulating influenza virus strains, all influence the serologic response to vaccination.However, even when these vaccines are administered to young fit adults a proportion of individuals do not mount a significant serologic response to the vaccine. It is not clear whether these nonresponding individuals are genetically pre-programmed to be nonresponders or whether failure to respond to the vaccine is a random event. There is good evidence that nonresponsiveness to hepatitis B vaccine, another purified protein vaccine, is at least partially modulated by an individual’s human leucocyte antigen (HLA) alleles. Because CD4+ T cells, which control the neutralizing antibody response to influenza virus, recognize antigens in association with HLA class II molecules, we recently conducted a small study to investigate whether there was any association between HLA class II molecules and nonresponsiveness to influenza virus vaccination. This work revealed that the HLA-DRB1*0701 allele was over represented among persons who fail to mount a neutralizing antibody response. This preliminary finding is important because it potentially identifies a group who may not be protected by current vaccination strategies. Further investigation into the role of HLA polymorphisms and nonresponse to influenza virus vaccination, and vaccination against viruses in general, is clearly required.

New millennium antivirals against pandemic and epidemic influenza: the neuraminidase inhibitors.

The mushroom shaped outer spike protein of influenza, neuraminidase, was first discovered nearly 60 years ago. Its importance in viral replication was soon recognised both at the point of viral release from the cell and also enabling passage of virus through nasal fluid to reach the cell. The enzyme active site was identified by x-ray crystallography, allowing an atomic study of interaction of enzyme with the sialic acid substrate. Analogues could then be identified and synthesized and became a focused target for antivirals. With the current threat of bioterrorism and the potential for the emergence of a new pandemic strain in the near future, efforts are underway to develop more potent second-generation anti-neuraminidase inhibitors with enhanced protective and therapeutic effects. Here we review older and newer neuraminidase inhibitors and the role that they will play in the fight against influenza in its epidemic and pandemic face.

A throat lozenge containing amyl meta cresol and dichlorobenzyl alcohol has a direct virucidal effect on respiratory syncytial virus, influenza A and SARS-CoV.

A potent virucidal mixture containing amyl metacresol and dichlorobenzyl alcohol at low pH inactivated enveloped respiratory viruses influenza A, respiratory synctial virus (RSV) and severe acute respiratory syndrome coronavirus (SARS-CoV) but not viruses with icosahedral symmetry, such as adenoviruses or rhinoviruses. A titre of approximately 3.5 log10 TCID50 was reduced to below the level of detection within two minutes. Electron microscopy of purified influenza A virus showed extensive clumping and morphological changes in spike configuration after contact with the virucidal mixture, but no overt destruction of the viral membrane. We conclude that, formulated as a lozenge, the mixture could have significant effects in reducing the infectivity of certain infectious viruses in the throat and presumably in cough droplets, thus reducing, theoretically, opportunities for person-to-person transmission.

New Antiviral Drugs, Vaccines and Classic Public Health Interventions against SARS Coronavirus

Severe acute respiratory syndrome (SARS) is caused by one of two recently discovered coronaviruses. The virus is emergent from South East (SE) Asian mammals: either the civet cat, a related species or a rat species. The virus has a long incubation period and low reproduction number (R0 value) and hence the first outbreak in 2004 was controlled by hygiene and quarantine. However, the healthcare system was compromised and the economic cost was extremely high. Fortunately, the virus is easily cultivated in Vero E6 cells and therefore the search for new antivirals and vaccines was initiated within weeks of the discovery of the virus using classic techniques of cell culture and electron microscopy. Molecular diagnostics facilitated rapid and accurate diagnosis, a key factor in containing the outbreak. The broad-spectrum molecule ribavirin was used in SE Asia in infected patients alongside corticosteroids. In retrospect, many patients survived due to careful nursing. The only currently accepted intervention is interferon. Coronavirus replicon systems should facilitate rapid screening of new inhibitors and the complex mechanism of viral replication will ensure that drugs are developed against at least five molecular targets, in particular the viral protease.

Preclinical in vitro activity of QR-435 against influenza A virus as a virucide and in paper masks for prevention of viral transmission.

Prophylaxis against influenza is difficult, and current approaches against pandemics may be ineffective because of shortages of the two proven classes of antivirals in the face of a large-scale infection. Herbal/natural products may represent an effective alternative to conventional attempts to protect against infection by avian influenza virus. QR-435, an all-natural compound of green tea extract and other agents, has been developed to provide protection against a wide range of viral infections. The antiviral activities of several QR-435 preparations as well as QR-435 (1) green tea extract were tested against A/Sydney/5/97 and A/Panama-Resvir 17 strains of avian influenza virus H3N2 by means of an assay based on Madin-Darby canine kidney cells. Toxic effects of QR-435 formulations on these cells were also evaluated as were the virucidal properties of a commercially available mask impregnated with QR-435. The efficacy of a QR-435/mask combination was compared with that of the QR control/mask combination, an untreated mask, and no mask. QR-435 had significant in vitro activity against H3N2 at concentrations that were not associated with significant cellular toxic effects. The antiviral activity of QR-435 (1) was similar to that of QR-435. Masks impregnated with QR-435 were highly effective in blocking the passage of live H3N2 virus. These preclinical results warrant further evaluation of the prophylactic use of QR-435 against viral infection in humans.

In vivo prophylactic activity of QR-435 against H3N2 influenza virus infection

BackgroundProphylaxis against influenza infection can take several forms, none of which is totally effective at preventing the spread of the disease. QR-435, an all-natural compound of green-tea extract and other agents, has been developed to protect against a range of viral infections, including the influenza subtype H3N2. MethodsSeveral different QR-435 formulations were tested against the two influenza A H3N2 viruses (A/Sydney/5/97 and A/Panama/2007/99) in the ferret model. Most experiments included negative (phosphate-buffered saline) and positive (oseltamivir 5 mg/kg, twice daily) controls. QR-435 and the control were administered 5 minutes after intranasal delivery of the virus as prophylaxis against infection resulting from exposure to infected but untreated ferrets and for prevention of transmission from infected and treated ferrets to untreated animals. Effects of QR-435 on seroconversion, virus shedding, and systemic sequelae of infection (weight loss, fever, reduced activity) were evaluated. ResultsQR-435 prevented transmission and provided prophylaxis against influenza virus H3N2. Prophylaxis with QR-435 was significantly more than with oseltamivir in these experiments. Optimal in vivo efficacy of QR-435 requires a horseradish concentration of at least 50% of that in the original formulation, and the benefits of this preparation appear to be dose dependent. ConclusionsQR-435 is effective for both prevention of H3N2 viral transmission and prophylaxis. These preclinical results warrant further evaluation of its prophylactic properties against avian influenza virus infection in humans.

Influenza — The Chameleon Virus

Publisher Summary Human influenza virus was first isolated and identified in the United Kingdom in 1933. The prerequisites for the work on the influenza virus were the discovery of a vital protein of the virus, hemagglutinin (HA); identification of the hemagglutinating ability of the influenza virus in chicken and mammalian erythrocytes; and, most importantly, discovery of the hemagglutination inhibition (HI) test. This chapter focuses on the HA protein of the influenza A virus. The first serological study in animal models and postinfection human sera indicated a surprising degree of antigenic change or drift of the HA. It was the emergence of a completely novel influenza A virus in Asia in 1957 and another in 1968 that confirmed the suspicion—harbored since the Great Spanish Pandemic of 1918—that the influenza A virus had two epidemiological faces or modes, epidemic and pandemic. With the advent of new techniques of protein chemistry and electron microscopy, it has become clear that the HA is the most important antigen of the virus and is made up of two polypeptides—HA1 and HA2. These two constituents of the HA were separated and studied by peptide mapping, and it was discovered that antigenic changes occur entirely in HAl molecules.

AIDS vaccine development: let a thousand flowers bloom.

Chemotherapy against HIV infection has progressed rapidly, with the almost immediate discovery of a partially eVective drug, azidothymidine (AZT), followed by the chemically related dideoxy nucleoside analogues ddI, ddC, 3TC, and more recently the viral protease inhibitors. 2 In comparison, scientific work towards development of an eVective vaccine against HIV has moved far more slowly and there are no such vaccines at present. This slower pace is not owing to any lack of eVort or ingenuity but rather to the diYcult task of preventing infection by an antigenically variable virus at a mucosal surface. However, influenza virus also falls into this category and yet the inactivated virus vaccine has proven eYcacy in the “at risk” groups, preventing hospital admission, serious illness and death. On the other hand most viral vaccines in current use such as measles, mumps, yellow fever, and influenza do not prevent an initial infection, but rather prevent an extension of that initial focus of infection. Herein lies a potential problem: HIV can integrate its genome into the genome of the infected cell, presumably at the initial infection as well as later. Could this viral property negate the eVect of any vaccine or is this only one of a number of “theoretical” worries which impede progress? It has also been argued that the development of a vaccine has not been helped by our incomplete or contradictory knowledge of AIDS immunopathology. Take, for example, one controversial hypothesis to explain the immunopathology of AIDS. Virologists tend to be comforted with explanations of high level of viral replication in CD4 cells leading to their demise and the collapse of the immune system. Immunologists believe in a more subtle viral strategy, of which the most controversial albeit compelling idea is that of autoimmunity combined with molecular mimicry—in essence that AIDS is an autoimmune disease triggered by the HIV virus, very much like graft versus host disease. A fragment of the viral spike glycoprotein (gp120) mimics a portion of the HLA DR (major histocompatibility complex (MHC) class II). This HLA region is hypervariable in the human population and in general governs the autoimmune and immune reaction. If by chance in a particular individual the structural similarities between this HLA fragment and the viral gp120 are very close then the virus will be recognised as self and there will be an autoimmune reaction to it. On the other hand, and more likely, the viral peptide, once presented in the groove of the HLA class I molecule, will be recognised as foreign; and the autoimmune reaction which would inevitably follow would destroy virus infected cells and also uninfected bystander CD4 cells as well. This hypothesis may well be highly relevant to AIDS vaccine development, as it may become necessary to delete this “autoimmune” peptide from the gp120 spike in a vaccine. However, a similar dearth of information (or contradictory information) did not retard other virus vaccines being manufactured and applied. Thus we are forced to ask serious questions about the slow progress towards an eVective AIDS vaccine. Pivotal in our opinion is whether too great and too diversified scientific eVort has been placed into “vaccine design,” using exciting discoveries in molecular biology. Has the pure scientific excitement diverted manpower and expertise from what is, or at least has been in the past, a very practical task? In reality only a small cohort of perhaps 10 000 scientists in the world is directly working on HIV vaccines. This would probably be ample to ensure the progress of one or two vaccine projects to fruition, but it is pitifully few to be working on dozens of novel ideas. Is HIV such a special pathogen that the old established approaches of killed virus or live attenuated virus vaccines would either not be expected to provide protection or be unsafe, and therefore futuristic molecular approaches are essential? We would argue that there are very few special features of HIV compared with other viruses, and so it need not be a special case. We are now into the second decade of the AIDS epidemic and the virus is showing ominous but predictable signs of evolving in new directions. To date, there have been two distinct epidemics. One occurs primarily in Europe and the USA and mainly aVects intravenous drug users and men who have sex with men. The second epidemic centres on Asia and Africa, where the virus is spread primarily among heterosexuals. There is some evidence that distinct viruses exist with varying ability to grow in rectal or vaginal cells, which is of course a worrying situation. Thus the need to develop an eVective HIV vaccine is now more urgent than ever. There are approximately 20 million individuals infected with HIV and 4.5 million AIDS cases worldwide. The African epidemic has cost over

Treatment of epidemic and pandemic influenza with neuraminidase and M2 proton channel inhibitors

30 billion and the United Nations estimates that AIDS will reduce Africa’s overall labour force by as much as 25% by the year 2010. At the current rate, the impact on the worldwide economy is estimated to reach