Peter D. Cooper

A genetic map of poliovirus temperature-sensitive mutants

Abstract The conditions affecting recombination between ts mutants of poliovirus and the assay of ts + recombinants were standardized. The mean recombinant frequencies were then reproducible and characteristic of each pair of mutants, being between 5.2 and 31.4 times the total background reversion rates and ranging from 0.02% to 0.85%. Certain genetic sequences were proved by three-factor crosses involving a mutant adapted to guanidine resistance ( ts-28 g ), and these showed that recombination frequencies were additive. An additive linear genetic map comprising one linkage group was obtained. The g character of ts-28 g seemed to result from mutation at a single site that could be accurately located near the middle of the map. Recombination was nonrandom with time, most of the mating events occurring early in the growth cycle.

Inulin-derived adjuvants efficiently promote both Th1 and Th2 immune responses

There has been a recent resurgence of interest into new and improved vaccine adjuvants. This interest has been stimulated by the need for new vaccines to combat problematic pathogens such as SARS and HIV, and to counter potential bioterrorist attacks. A major bottleneck in vaccine development is the low immunogenicity of purified subunit or recombinant proteins, creating the need for safe human adjuvants with high potency. A major problem in the search for the ideal adjuvant is that adjuvants that promote cell‐mediated (Th1) immunity (e.g. Freunds complete adjuvant) generally have unacceptable local or systemic toxicity that precludes their use in human vaccines. There is a need for a safe, non‐toxic adjuvant that is able to stimulate both cell‐mediated and humoral immunity. Inulin‐derived adjuvants that principally stimulate the innate immune system through their ability to activate the alternative complement pathway have proven ability to induce both cellular and humoral immunity. With their excellent tolerability, long shelf‐life, low cost and easy manufacture, they offer great potential for use in a broad range of prophylactic and therapeutic vaccines. Based on successful animal studies in a broad range of species, human trials are about to get underway to validate the use of inulin‐based adjuvants in prophylactic vaccines against hepatitis B, malaria and other pathogens. If such trials are successful, then it is possible that inulin‐derived adjuvants will one day replace alum as the adjuvant of choice in most human prophylactic vaccines.

An Improved Agar Cell-Suspension Plaque Assay for Poliovirus : some Factors affecting Efficiency of Plating.

Abstract A method is given for a convenient agar cell-suspension plaque assay of poliovirus using the ERK transformed cell line, and avoiding the need for a CO 2 -bicarbonate or other additional buffer by using galactose in place of glucose. A number of factors increased the sensitivity four- to sixfold and made the assay more uniform.

An inactivated Vero cell-grown Japanese encephalitis vaccine formulated with Advax, a novel inulin-based adjuvant, induces protective neutralizing antibody against homologous and heterologous flaviviruses

Advax is a polysaccharide-based adjuvant that potently stimulates vaccine immunogenicity without the increased reactogenicity seen with other adjuvants. This study investigated the immunogenicity of a novel Advax-adjuvanted Vero cell culture candidate vaccine against Japanese encephalitis virus (JEV) in mice and horses. The results showed that, in mice, a two-immunization, low-dose (50 ng JEV antigen) regimen with adjuvanted vaccine produced solid neutralizing immunity comparable to that elicited with live ChimeriVax-JE immunization and superior to that elicited with tenfold higher doses of a traditional non-adjuvanted JEV vaccine (JE-VAX; Biken Institute) or a newly approved alum-adjuvanted vaccine (Jespect; Novartis). Mice vaccinated with the Advax-adjuvanted, but not the unadjuvanted vaccine, were protected against live JEV challenge. Equine immunizations against JEV with Advax-formulated vaccine similarly showed enhanced vaccine immunogenicity, confirming that the adjuvant effects of Advax are not restricted to rodent models. Advax-adjuvanted JEV vaccine elicited a balanced T-helper 1 (Th1)/Th2 immune response against JEV with protective levels of cross-neutralizing antibody against other viruses belonging to the JEV serocomplex, including Murray Valley encephalitis virus (MVEV). The adjuvanted JEV vaccine was well tolerated with minimal reactogenicity and no systemic toxicity in immunized animals. The cessation of manufacture of traditional mouse brain-derived unadjuvanted JEV vaccine in Japan has resulted in a JEV vaccine shortage internationally. There is also an ongoing lack of human vaccines against other JEV serocomplex flaviviruses, such as MVEV, making this adjuvanted, cell culture-grown JEV vaccine a promising candidate to address both needs with one vaccine.

Carbohydrate-based immune adjuvants

The role for adjuvants in human vaccines has been a matter of vigorous scientific debate, with the field hindered by the fact that for over 80 years, aluminum salts were the only adjuvants approved for human use. To this day, alum-based adjuvants, alone or combined with additional immune activators, remain the only adjuvants approved for use in the USA. This situation has not been helped by the fact that the mechanism of action of most adjuvants has been poorly understood. A relative lack of resources and funding for adjuvant development has only helped to maintain alum’s relative monopoly. To seriously challenge alum’s supremacy a new adjuvant has many major hurdles to overcome, not least being alum’s simplicity, tolerability, safety record and minimal cost. Carbohydrate structures play critical roles in immune system function and carbohydrates also have the virtue of a strong safety and tolerability record. A number of carbohydrate compounds from plant, bacterial, yeast and synthetic sources have emerged as promising vaccine adjuvant candidates. Carbohydrates are readily biodegradable and therefore unlikely to cause problems of long-term tissue deposits seen with alum adjuvants. Above all, the Holy Grail of human adjuvant development is to identify a compound that combines potent vaccine enhancement with maximum tolerability and safety. This has proved to be a tough challenge for many adjuvant contenders. Nevertheless, carbohydrate-based compounds have many favorable properties that could place them in a unique position to challenge alum’s monopoly over human vaccine usage.

Genetics of Picornaviruses

Picornaviruses have long seemed to have many genes. The size of the genome compared with the “average” gene product suggested about ten for poliovirus (Fenner, 1968), which causes at least a dozen synthetic and degenerative changes in the host cell. Many viral polypeptides are found, originally about 14 (Summers et al.,1965) and now more than 34 (Abraham and Cooper, 1975a). Other picornaviruses are very similar.

Delta inulin: a novel, immunologically active, stable packing structure comprising β-d-[2 → 1] poly(fructo-furanosyl) α-d-glucose polymers

We report a novel isoform of β-D-[2 → 1] poly(fructo-furanosyl) α-D-glucose termed delta inulin (DI), comparing it with previously described alpha (AI), beta (BI) and gamma (GI) isoforms. In vitro, DI is the most immunologically active weight/weight in human complement activation and in binding to monocytes and regulating their chemokine production and cell surface protein expression. In vivo, this translates into potent immune adjuvant activity, enhancing humoral and cellular responses against co-administered antigens. As a biocompatible polysaccharide particle, DI is safe and well tolerated by subcutaneous or intramuscular injection. Physico-chemically, DI forms as an insoluble precipitate from an aqueous solution of suitable AI, BI or GI held at 37-48°C, whereas the precipitate from the same solution at lower temperatures has the properties of AI or GI. DI can also be produced by heat conversion of GI suspensions at 56°C, whereas GI is converted from AI at 45°C. DI is distinguished from GI by its higher temperature of solution in dilute aqueous suspension and by its lower solubility in dimethyl sulfoxide, both consistent with greater hydrogen bonding in DIs polymer packing structure. DI suspensions can be dissolved by heat, re-precipitated by cooling as AI and finally re-converted back to DI by repeated heat treatment. Thus, DI, like the previously described inulin isoforms, reflects the formation of a distinct polymer aggregate packing structure via reversible noncovalent bonding. DI forms the basis for a potent new human vaccine adjuvant and further swells the growing family of carbohydrate structures with immunological activity.

THE MUTATION OF POLIOVIRUS BY 5-FLUOROURACIL.

Abstract Poliovirus grown in 4 m M 5-fluorouracil (5-FU) at an optimum temperature (36°) becomes more “heat defective”; that is, the ratio of plaques formed at supraoptimal temperatures to plaques formed at 36° (“plating efficiency”) is decreased by a factor of 3–10. The plating efficiency at an infraoptimal temperature (30°) is unchanged. These findings are true for strains with very different plating efficiencies at both high and low temperatures. The enhanced heat defectiveness is retained after subculture in absence of 5-FU: since selection of preexisting heat-defective ( hd ) mutants is shown to be unlikely, it is concluded that 5-FU is highly mutagenic for poliovirus when judged by the character of plating efficiency at high temperatures. This is confirmed by the plaque isolation of hd mutants, which comprise at least 10% of the progeny of a nondefective ( hd + ) strain grown in 1 m M 5-FU; normal hd + progeny contain 1–2% of hd mutants. Poliovirus growth is inhibited by 5-FU to an extent which depends on the intrinsic heat defectiveness of the strain: the lower the plating efficiency at high temperatures, the poorer the growth in 5-FU at 36°.

Observation of the keto tautomer of d-fructose in D2O using 1H NMR spectroscopy

D-Fructose was analysed by NMR spectroscopy and previously unidentified (1)H NMR resonances were assigned to the keto and α-pyranose tautomers. The full assignment of shifts for the various fructose tautomers enabled the use of (1)H NMR spectroscopy in studies of the mutarotation (5-25°C) and tautomeric composition at equilibrium (5-50°C). The mutarotation of β-pyranose to furanose tautomers in D(2)O at a concentration of 0.18 M was found to have an activation energy of 62.6 kJmol(-1). At tautomeric equilibrium (20°C in D(2)O) the distribution of the β-pyranose, β-furanose, α-furanose, α-pyranose and the keto tautomers was found to be 68.23%, 22.35%, 6.24%, 2.67% and 0.50%, respectively. This tautomeric composition was not significantly affected by varying concentrations between 0.089 and 0.36 M or acidification to pH 3. Upon equilibrating at 6 temperatures between 5 and 50°C there was a linear relationship between the change in concentration and temperature for all forms.

The adjuvanticity of gamma inulin.

Gamma‐inulin (g‐IN) is a polymorph identified as the active component of inulin preparations that specifically activates the alternative pathway of complement (APC). The APC is central to many leucocyte functions, including B cell activation. We show here that g‐IN, when formulated as a pure, endotoxin‐free, fine suspension insoluble at 37°C and given at 50–100 μg per mouse, is a potent adjuvant for both humoral and cell‐mediated responses to a variety of antigens. g‐IN increased secondary IgG responses five‐ to 28‐fold (P <0·001), using as antigen phosphorylcholine coupled to keyhole limpet haemocyanin; subclasses IgG 2a, 2b, and 3 were boosted several hundred‐fold, IgG 1 10‐fold. IgM and IgA were increased four‐to six‐fold. Delayed hypersensitivity, by footpad swelling after secondary challenge with sheep red blood cells (SRBC), was increased more than two‐fold (P <0·001) if g‐IN was included with the primary SRBC, equivalent to increasing primary doses 10‐fold, g‐IN was equally active if given 5 days before the primary SRBC. Thus it is an immune stimulant rather than a depot or vehicle for antigen. Mice primed subcutaneously with 30–300 HA units of H2N2 influenza virus (strain A/JAP) and challenged intranasally with a lethal dose of HIN1 virus (strain A/WSN) all died, but if g‐IN was given with the primary antigen 50% of the mice survived(P < 0·001), a deduced but not proven boost to cytotoxic T cell‐mediated immunity. Unpublished work has shown that g‐IN has no adverse effects at adjuvant‐active doses. g‐IN is thus a promising new vaccine adjuvant. It also has a potential for antitumour therapy, and is a specific reagent for expioring the role of complement in vivo.