A Brief History of Human Challenge Studies (1900-2021) Emphasising the Virology, Regulatory and Ethical Requirements, Raison D'etre, Ethnography, Selection of Volunteers and Unit Design.

Venetian quarantine 400 years ago was an important public health measure. Since 1900 this has been refined to include "challenge" or deliberate infection with pathogens be they viruses, bacteria, or parasites. Our focus is virology and ranges from the early experiments in Cuba with Yellow Fever Virus to the most widespread pathogen of our current times, COVID-19. The latter has so far caused over four million deaths worldwide and 190 million cases of the disease. Quarantine and challenge were also used to investigate the Spanish Influenza of 1918 which caused over 100 million deaths. We consider here the merits of the approach, that is the speeding up of knowledge in a practical sense leading to the more rapid licensing of vaccines and antimicrobials. At the core of quarantine and challenge initiatives is the design of the unit to allow safe confinement of the pathogen and protection of the staff. Most important though is the safety of volunteers. We can see now, as in 1900, that members of our society are prepared and willing to engage in these experiments for the public good. Our ethnology study, where the investigator observed the experiment from within the quarantine, gave us the first indication of changing attitudes amongst volunteers whilst in quarantine. These quarantine experiments, referred to as challenge studies, human infection studies, or "controlled human infection models" involve thousands of clinical samples taken over two to three weeks and can provide a wealth of immunological and molecular data on the infection itself and could allow the discovery of new targets for vaccines and therapeutics. The Yellow Fever studies from 121 years ago gave the impetus for development of a successful vaccine still used today whilst also uncovering the nature of the Yellow Fever agent, namely that it was a virus. We outline how carefully these experiments are approached and the necessity to have high quality units with self-contained air-flow along with extensive personal protective equipment for nursing and medical staff. Most important is the employment of highly trained scientific, medical and nursing staff. We face a future of emerging pathogens driven by the increasing global population, deforestation, climate change, antibiotic resistance and increased global travel. These emerging pathogens may be pathogens we currently are not aware of or have not caused outbreaks historically but could also be mutated forms of known pathogens including viruses such as influenza (H7N9, H5N1 etc.) and coronaviruses. This calls for challenge studies to be part of future pandemic preparedness as an additional tool to assist with the rapid development of broad-spectrum antimicrobials, immunomodulators and new vaccines.

Recovery of stem cell proliferation by low intensity vibration under simulated microgravity requires LINC complex.

Mesenchymal stem cells (MSC) rely on their ability to integrate physical and spatial signals at load bearing sites to replace and renew musculoskeletal tissues. Designed to mimic unloading experienced during spaceflight, preclinical unloading and simulated microgravity models show that alteration of gravitational loading limits proliferative activity of stem cells. Emerging evidence indicates that this loss of proliferation may be linked to loss of cellular cytoskeleton and contractility. Low intensity vibration (LIV) is an exercise mimetic that promotes proliferation and differentiation of MSCs by enhancing cell structure. Here, we asked whether application of LIV could restore the reduced proliferative capacity seen in MSCs that are subjected to simulated microgravity. We found that simulated microgravity (sMG) decreased cell proliferation and simultaneously compromised cell structure. These changes included increased nuclear height, disorganized apical F-actin structure, reduced expression, and protein levels of nuclear lamina elements LaminA/C LaminB1 as well as linker of nucleoskeleton and cytoskeleton (LINC) complex elements Sun-2 and Nesprin-2. Application of LIV restored cell proliferation and nuclear proteins LaminA/C and Sun-2. An intact LINC function was required for LIV effect; disabling LINC functionality via co-depletion of Sun-1, and Sun-2 prevented rescue of cell proliferation by LIV. Our findings show that sMG alters nuclear structure and leads to decreased cell proliferation, but does not diminish LINC complex mediated mechanosensitivity, suggesting LIV as a potential candidate to combat sMG-induced proliferation loss.

Choosing sample sizes for various blood parameters of broiler chickens with normal and non-normal observations.

Experimental power is a measure of the ability of an experiment to detect differences between treatment means. Researchers design experiments and then calculate the probability that differences are simply due to chance, the null hypothesis. The objective of the analyses reported here was to determine the appropriate number of samples to demonstrate significant differences of various magnitudes from broiler chicken blood constituents. Over 800 samples were taken for a study of the effects of sample storage time, serum vs. plasma, light intensity, and fed vs. fasted birds on blood cholesterol, triglycerides, uric acid, glucose, total protein (TP), albumin, globulin, alanine aminotransferase (ALT), aspartate aminotransferase, gammaGT, creatinine, alkaline phosphatase, Ca and P. Various transformations increased the QQ plot R2 values from 0.000 to 0.149 or 0.00 to 17.62%. Most of the QQ plot R2 values were at or above 0.90. The 1/x2 transformation of blood P data showed the biggest increase in QQ plot R2 (0.846 to 0.995). The different standard deviations and coefficients of variation (CVs) found for each variable resulted in widely different numbers of replicates needed to detect differences in 2 treatment means. The extremes were glucose with a CV of 6.9% and ALT with a CV of 39.7%. For glucose, 15 replicates are needed to find a 10% difference in 97% of experiments; for ALT, 15 replicates would detect a 50% difference 91% of the time. The use of parameters such as cholesterol, glucose, TP, albumin, and globulin showed low CVs, indicating they may be considered as stable parameters. The lower CVs make it possible to find differences with a smaller number of replicates used in studies. As reported, the phosphorus values did not have a normal distribution of the data, so a transformation of these data could be an alternative to better discuss the results found.

A review of nanoparticle functionality and toxicity on the central nervous system.

Although nanoparticles have tremendous potential for a host of applications, their adverse effects on living cells have raised serious concerns recently for their use in the healthcare and consumer sectors. As regards the central nervous system (CNS), research data on nanoparticle interaction with neurons has provided evidence of both negative and positive effects. Maximal application dosage of nanoparticles in materials to provide applications such as antibacterial and antiviral functions is approximately 0.1-1.0 wt%. This concentration can be converted into a liquid phase release rate (leaching rate) depending upon the host or base materials used. For example, nanoparticulate silver (Ag) or copper oxide (CuO)-filled epoxy resin demonstrates much reduced release of the metal ions (Ag(+) or Cu(2+)) into their surrounding environment unless they are mechanically removed or aggravated. Subsequent to leaching effects and entry into living systems, nanoparticles can also cross through many other barriers, such as skin and the blood-brain barrier (BBB), and may also reach bodily organs. In such cases, their concentration or dosage in body fluids is considered to be well below the maximum drug toxicity test limit (10(-5) g ml(-1)) as determined in artificial cerebrospinal solution. As this is a rapidly evolving area and the use of such materials will continue to mature, so will their exposure to members of society. Hence, neurologists have equal interests in nanoparticle effects (positive functionality and negative toxicity) on human neuronal cells within the CNS, where the current research in this field will be highlighted and reviewed.

The end of the beginning: vaccines for the next 25 years.

The first virus vaccines against smallpox and rabies proved their effectiveness even before the ultra microscopic viruses had been identified as a new world of infectious agents. To date most antibacterial and antiviral vaccines have not been designed but rather built step by step. Designer vaccines with T cell epitopes and adjuvants which stimulate innate or acquired immune responses to will are now under serious investigation but have yet to impact on the practical world of infection. The latter is not small, with millions of deaths annually in the world from not uncommon microbes such as enterforms, pneumococci, respiratory and hepatitis viruses and HIV. But can vaccines be used in more social directions to control birth or prevent addiction? Polio should join smallpox this year in the pantheon of eradicated viruses. The infectious disease community can then turn attention to hepatitis B. War has been declared on pandemic influenza but with this zoonotic virus containment is key, with vaccines used alongside antivirals and social distancing. Undoubtedly "we have the guns, and now we can finish the job".

Standardized, mathematical model-based and validated in vitro analysis of anthrax lethal toxin neutralization.

Quantification of anthrax lethal toxin (LTx) neutralization activity (TNA) is pivotal in assessing protective antibody responses to anthrax vaccines and for evaluation of immunotherapies for anthrax. We have adapted and redesigned the TNA assay to establish a unifying, standardized, quantitative and validated technology platform for LTx neutralization in the J774A.1 murine cell line. Critical design features of this platform are 1) the application of a free-form or constrained 4 parameter logistic (4-PL) function to model neutralization responses within and between boundary limits of 100% cell survival and 95% cell lysis and 2) to exploit innovative assay curve recognition algorithms for interpretive endpoints. The assay was validated using human serum ED50 (dilution of serum effecting 50% neutralization) as the primary reportable value (RV). Intra-operator and intermediate precision, expressed as the coefficient of variation (%CV), were high at 10.5-15.5%CV and 13.5-14.5%CV respectively. TNA assay dilutional linearity was demonstrated for human sera using linear regression analysis of log(10) transformed data with slope=0.99, intercept=-0.03 and r(2)=0.985. Assay accuracy, inferred from the precision and linearity data and using a spike-recovery approach, was high with a percent error (%E) range of only 3.4-20.5%E. The lower limit of detection (LLOD) was ED50=12 and the lower limit of quantification (LLOQ) was ED50=36. The cell-based assay was robust, tolerating incubation temperatures from 35 to 39 degrees C, CO(2) concentrations from 3% to 7% and reporter substrate (MTT) concentrations of 2.5-7.5 mg/ml. Strict assay quality control parameters were met for up to 25 cell culture passages. The long term (50 month) assay stability, determined using human reference standards AVR414 and AVR801, indicated high precision, consistent accuracy and no detectable assay drift. A customized software program provided two additional assay metrics, Quantification Titer (QT) and Threshold Titer (TT), both of which demonstrate acceptable accuracy, precision and dilutional linearity. The TT was also used to establish the assay reactivity threshold (RT). The application of the assay to sera from humans, Rhesus macaques and rabbits was demonstrated separately and by aggregate dilutional linearity analysis of the ED50 (slope=0.98, intercept=0.003, r(2)=0.989). We propose this TNA assay format with a qualified standard reference serum and customized interpretive software as a unifying platform technology for determination of functional serologic responses to anthrax vaccines and for evaluation of anthrax immunotherapeutics.

Scientific lessons from the first influenza pandemic of the 20th century.

Re-analysis of the influenza pandemic of 1918 has given reassurance about a rather low reproductive number (R(o)), a prolonged herald wave of virus and that the skewed mortality towards the young adult could be a singularly unique event dependent upon previous infection history, perhaps not to be repeated in a future pandemic. Over 99% of those who contracted the virus survived, in spite of the absence of antivirals, vaccine and antibiotics for the secondary bacteria infections which probably accounted for one-third of the 50 million deaths. Therefore, in spite of a three-fold population increase since 1918 and 100 thousand plane journeys daily, judicious and careful planning together with a stockpile of antiviral drugs, oseltamivir, zanamivir and M2 blockers and a generic H5N1 vaccine, and application of hygiene would be expected to reduce mortality in a new pandemic, to figures significantly less than 1918.

Interfering vaccine (defective interfering influenza A virus) protects ferrets from influenza, and allows them to develop solid immunity to reinfection.

Defective interfering (DI) virus RNAs result from major deletions in full-length viral RNAs that occur spontaneously during de novo RNA synthesis. These RNAs are packaged into virions that are by definition non-infectious, and are delivered to cells normally targeted by the virion. DI RNAs can only replicate with the aid of a coinfecting infectious helper virus, but the small size of DI RNA allows more copies of it to be made than of its full-length counterpart, so the cell produces defective virions in place of infectious progeny. In line with this scenario, the expected lethal disease in an influenza A virus-mouse model is made subclinical by administration of DI virus, but animals develop solid immunity to the infecting virus. Hence DI virus has been called an 'interfering vaccine'. Because interfering vaccine acts intracellularly and at a molecular level, it should be effective against all influenza A viruses regardless of subtype. Here we have used the ferret, widely acknowledged as the best model for human influenza. We show that an interfering vaccine with defective RNAs from an H3N8 virus almost completely abolished clinical disease caused by A/Sydney/5/97 (H3N2), with abrogation of fever and significant reductions in clinical signs of illness. Animals recovered fully and were solidly immune to reinfection, in line with the view that treatment converts the otherwise virulent disease into a subclinical and immunizing infection.

Influenza is now a preventable disease.

The world is waiting with apprehension for the predicted pandemic of H5N1 (avian) influenza as an increasing number of countries in Asia, Europe and Africa report cases of influenza in migrating birds. All is not 'despondency', however. Targeted and controlled administration of antiviral drugs, alone or in combination, to contacts and cases, together with well tried public health measures, should slow down the spread of the infection and allow time for vaccines to be developed, thus preventing a worldwide pandemic of the type that occurred in 1918.

A new European perspective of influenza pandemic planning with a particular focus on the role of mammalian cell culture vaccines.

Sixteen EU scientists and doctors were interviewed about pandemic planning using psychometric methods applied to a scientific problem for the first time. Criticism was aimed at countries which have no plan whatsoever, the majority of nations. Many such countries have not invested in scientific infrastructure and public health. Amongst the 15 or so published pandemic plans a lack of detail was identified. Of particular need was investment into avian virus vaccine stocks (H1-15), prepared licenses of vaccine and pre purchase and agreed distribution, investment into stocks of antivirals, antibiotics and masks. Most but not all members of the group predicted a global outbreak within 5 years, most probably starting in SE Asia. However it was recognised that a pandemic could start anywhere in the world which had juxtaposition of young people, chickens, ducks and pigs. Mammalian cell culture production using wild type virus with the production factory at category III levels of security was exemplified. Antivirals would be essential to ameliorate the first wave of infection although significant quantities of cell grown vaccine could be produced if, as in 1918, 1957 and 1968 there is a long period between the first virus isolation and person to person spread. The wider scientific community is more energised than previously for very serious preparations to be in place way before the outbreak begins as this is a major public health problem, completely dwarfing concerns about bioterrorism.

A hypothesis: the conjunction of soldiers, gas, pigs, ducks, geese and horses in northern France during the Great War provided the conditions for the emergence of the "Spanish" influenza pandemic of 1918-1919.

The Great Influenza Pandemic of 1918-1919 was a cataclysmic outbreak of infection wherein over 50 million people died worldwide within 18 months. The question of the origin is important because most influenza surveillance at present is focussed on S.E. Asia. Two later pandemic viruses in 1957 and 1968 arose in this region. However we present evidence that early outbreaks of a new disease with rapid onset and spreadability, high mortality in young soldiers in the British base camp at Etaples in Northern France in the winter of 1917 is, at least to date, the most likely focus of origin of the pandemic. Pathologists working at Etaples and Aldershot barracks later agreed that these early outbreaks in army camps were the same disease as the infection wave of influenza in 1918. The Etaples camp had the necessary mixture of factors for emergence of pandemic influenza including overcrowding (with 100,000 soldiers daily changing), live pigs, and nearby live geese, duck and chicken markets, horses and an additional factor 24 gases (some of them mutagenic) used in large 100 ton quantities to contaminate soldiers and the landscape. The final trigger for the ensuing pandemic was the return of millions of soldiers to their homelands around the entire world in the autumn of 1918.

Lack of detection of influenza genes in archived formalin-fixed, paraffin wax-embedded brain samples of encephalitis lethargica patients from 1916 to 1920.

A method was developed for detection of influenza genes in formalin-fixed brains of mice that had been experimentally infected with influenza A/NWS/33 (H1N1) virus. Using this technique, messenger ribonucleic acid (mRNA) of the beta-actin gene was detected in eight clinical brain samples from the 1916-1920 outbreak of encephalitis lethargica, showing preservation of particular mRNAs. However, we did not detect influenza nucleotide sequences of M, NP, and NS genes from these same samples. We conclude either that influenza was not the causative agent of encephalitis lethargica or, possibly, that the virus had a hit-and-run mechanism and was no longer present in the brain at the time of death of the patients.

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

A small armentarium of anti-influenza drugs now exists, and includes the M2 blockers (amantadine and rimantadine) and the neuraminidase inhibitors (Relenza and Tamiflu). The neuraminidase inhibitors have certain advantages, including a broader spectrum of antiviral activity, including influenza A and B viruses. On the other hand, there is now much clinical experience with the M2 blockers, and these drugs are inexpensive. It is clear that influenza in different community groups needs to be managed in specific and targeted ways. For example, in the over-65-years and at-risk groups, vaccination will remain a mainstay of disease prevention. However, up to 40% of those in these groups may fail to receive vaccine, and therefore the antivirals can be used therapeutically, or, in defined circumstances, as prophylactics. At present, influenza is hardly managed in the community. The infrequent global outbreaks, pandemics, present further problems. The more extensive use of the two classes of antivirals, and also vaccines, in the important interpandemic years will provide a very significant investment in health benefits in the face of a new pandemic virus in an otherwise completely vulnerable population.

A designer drug against influenza: the NA inhibitor oseltamivir (Tamiflu).

The description of the first two designer antiviral drugs to fight influenza was a ground breaking advance. Targeted against the influenza neuraminidase enzyme these inhibitors have been shown to reduce both the severity and duration of influenza illness. Importantly, it is expected that these neuraminidase inhibitors would be effective against influenza pandemic strain and could therefore be vital at reducing the potentially devastating consequences of such an outbreak. Despite the demonstrated efficacy of these drugs, they are not commonly used, particularly in the UK, and there is substantial concern that in the event of a pandemic or even a severe epidemic there could be substantial morbidity and mortality. SARS has shown that the public and media response to a serious epidemic is not always rational and this could easily become panic if it became apparent that treatment was possible, but not available.

The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo.

Oseltamivir carboxylate is a potent and specific inhibitor of influenza A and B neuraminidase (NA). Oseltamivir phosphate, the ethyl ester prodrug of oseltamivir carboxylate, is the first orally active NA inhibitor available for the prophylaxis and treatment of influenza A and B. It offers an improvement over amantadine and rimantadine which are active only against influenza A and rapidly generate resistant virus. The emergence of virus resistant to oseltamivir carboxylate in the treatment of naturally acquired influenza infection is low (about 1%). The types of NA mutation to arise are sub-type specific and largely predicted from in vitro drug selection studies. A substitution of the conserved histidine at position 274 for tyrosine in the NA active site has been selected via site directed mutagenesis, serial passage in culture under drug pressure in H1N1 and during the treatment of experimental H1N1 infection in man. Virus carrying H274Y NA enzyme selected in vivo has reduced sensitivity to oseltamivir carboxylate. The replicative ability in cell culture was reduced up to 3 logs, as was infectivity in animal models of influenza virus infection. Additionally, pathogenicity of the mutant virus is significantly compromised in ferret, compared to the corresponding wild type virus. Virus carrying a H274Y mutation is unlikely to be of clinical consequence in man.

Influenza virus carrying neuraminidase with reduced sensitivity to oseltamivir carboxylate has altered properties in vitro and is compromised for infectivity and replicative ability in vivo.

Oseltamivir phosphate (Tamiflu, Ro 64-0796) is the first orally administered neuraminidase (NA) inhibitor approved for use in treatment and prevention of influenza virus infection in man. Oseltamivir phosphate is the pro-drug of the active metabolite oseltamivir carboxylate (Ro 64-0802). Extensive monitoring throughout the oseltamivir development programme has identified a very low incidence of patients who have carried drug-resistant virus. The predominant mutation seen is the substitution of arginine for lysine at position 292 of the viral NA. The fitness of clinically isolated influenza virus A/Sydney/5/97 (H3N2) carrying this mutation was markedly reduced in animal models of influenza virus infection. The infectivity and replicative abilities of R292K mutant virus were reduced by at least 2 logs in a mouse model of influenza infection and by 2 and 4 logs, respectively, in the ferret model. Pathogenicity of R292K influenza virus A/Sydney/5/97 was reduced in ferrets as measured by inflammatory and febrile responses at least in parallel to the decrease in replicative ability. The data indicate that the R292K NA mutation compromises viral fitness such that virus carrying this mutation is unlikely to be of significant clinical consequence in man.