Hisashi Yamasaki

Arginine Facilitates Inactivation of Enveloped Viruses

Virus inactivation is a key step for the purification of pharmaceutical proteins derived from recombinant mammalian expression systems and conventionally done using low pH-treatment, which is often harmful to the proteins to be purified. This is particularly true for antibodies, because immunoglobulin proteins undergo conformational changes at acidic pH. We have been developing mild elution solvents using arginine for Protein-A chromatography to minimize the low pH-induced damages on the antibodies. Here we have tested the aqueous solutions containing arginine or butyroyl-arginine at or above pH 4.0 for their effects on virus inactivation, since these solvents are effective above pH 4.0 in elution of bound antibodies from Protein-A columns. When the virus was incubated on ice, 0.1 M sodium citrate was totally ineffective above pH 4.0, but aqueous solutions containing arginine above 0.35 M or butyroyl-arginine above 0.28 M showed extensive virus killing at or even above pH 4.0.

Inhibition of multiplication of herpes simplex virus by caffeic acid

Hot water extracts of coffee grinds and commercial instant coffee solutions have been shown to exhibit marked antiviral and virucidal activities against herpes simplex virus type 1 (HSV-1). Specifically, it has been shown that caffeine and N-methyl-pyridinium formate inhibit the multiplication of HSV-1 in HEp-2 cells. The present study examined the virological properties and the antiviral activity of caffeic acid against HSV-1. Caffeic acid inhibited the multiplication of HSV-1 in vitro, while chlorogenic acid, a caffeic acid ester with quinic acid, did not. These reagents did not have a direct virucidal effect. The one-step growth curve of HSV-1 showed that the addition of caffeic acid at 8 h post infection (h p.i.) did not significantly affect the formation of progeny viruses. An analysis of the influence of the time of caffeic acid addition, revealed that addition at an early time post infection remarkably inhibited the formation of progeny infectious virus in the infected cells, but its addition after 6 h p.i. (i.e., the time of the completion of viral genome replication) did not efficiently inhibit this process. These results indicate that caffeic acid inhibits HSV-1 multiplication mainly before the completion of viral DNA replication, but not thereafter. Although caffeic acid showed some cytotoxicity by prolonged incubation, the observed antiviral activity is likely not the secondary result of the cytotoxic effect of the reagent, because the inhibition of the virus multiplication was observed before appearance of the notable cytotoxicity.

Co-operative thermal inactivation of herpes simplex virus and influenza virus by arginine and NaCl.

Elevated temperatures have been used to inactivate viruses for plasma-derived biopharmaceuticals. This paper describes the effects of arginine and NaCl in conjunction with elevated temperature for inactivation of two enveloped viruses, i.e., herpes simplex virus type 1 (HSV-1) and influenza virus type A at neutral pH. In phosphate-buffered saline, a significant inactivation of HSV-1 occurred above 40 degrees C, resulting in less than 10% surviving virus (over 90% virus inactivation) at 50 degrees C. Arginine concentration dependently decreased the temperature required for virus inactivation, leading to temperature shift by almost 17 degrees C at 1.2M. NaCl also decreased the inactivation temperature, but to a considerably lesser extent, indicating that virus inactivation effect of arginine is not simply due to ionic strength. Influenza virus was also inactivated by high temperature, but its responses to arginine and NaCl were different from those on HSV-1, suggesting that virus inactivation mechanism is different between these two viruses, i.e., the effects of these reagents are virus specific.

Butyroyl-arginine as a potent virus inactivation agent.

Virus inactivation is a critical step in the manufacturing of recombinant therapeutic proteins, in particular antibodies, using mammalian expression systems. We have shown in the previous paper that arginine is effective in inactivation of herpes simplex virus type 1 (HSV-1) and influenza virus at low temperature under mildly acidic pH, i.e., above pH 4.0; above this pH, conformational changes of most antibodies are negligible. We have here extended virus inactivation study of arginine to other enveloped viruses, such as Sendai virus and Newcastle Disease Virus (NDV), and observed that arginine was ineffective against both viruses under the similar conditions, i.e., on ice and above pH 4.0. However, an arginine derivative, butyroyl-arginine, showed a strong virucidal potency against Sendai virus, leading to a 4log reduction in virus yield at pH 4.0, but not against NDV. In addition, although arginine and butyroyl-arginine were equally effective against influenza virus having a cleaved form of hemagglutinin spike proteins, only butyroyl-arginine was significantly effective against the same virus, but having an uncleaved hemagglutinin spike proteins. Furthermore, butyroyl-arginine was more effective than arginine against HSV-1 at pH 4.5; i.e., it has a broader pH spectrum than does arginine.

Antiviral and Virucidal Activities of Natural Products

Virus infection is one of the major threats to human health and can be avoided by minimizing exposure to infectious viruses. Viral clearance of pharmaceutical products and sanitization of skin and mucosal surfaces would reduce such exposures. Even with such care, virus infection does occur, requiring effective treatments by antiviral or virucidal agents. Natural products, in particular ingredients of foods and drinks we normally consume or metabolites present in human body at low concentrations, would have advantage over synthetic drugs as antiviral agents for safety concerns. For this reason, we have been studying natural products for their effects on virus inactivation and growth. Such natural products, which we have been focusing, include gallate derivatives, caffeine present in coffee, caffeic acid present in coffee and various fruits, ascorbic and dehydroascorbic acids and a cell metabolite, arginine. Here we will review our work on antiviral and virucidal activities of these compounds and the mechanism of their antiviral and virucidal effects.

Antiviral activities of coffee extracts in vitro.

Both hot water extracts of coffee grinds and instant coffee solutions inhibited the multiplication of herpes simplex virus type 1, a representative enveloped DNA virus, when they were added to the culture medium of the virus-infected cells at a dose of one fifth the concentration suitable for drinking. The antiherpetic activity was independent of the suppliers (companies) of the coffee grinds and of the locations where the coffee beans were produced. Further characterization revealed that there are two different mechanisms, by which the coffee extracts exert inhibitory activities on the virus infection; (1) a direct inactivation of the infectivity of virus particle (i.e., a virucidal activity) and (2) the inhibition of progeny infectious virus formation at the late stage of viral multiplication in the infected cells. Caffeine, but not quinic acid and chlorogenic acid, inhibited the virus multiplication to some extent, but none of them showed the virucidal activity, suggesting that other component(s) in the coffee extracts must play a role in the observed antiviral activity. In addition, the coffee extracts inhibited the multiplication of poliovirus, a non-enveloped RNA virus, but showed no virucidal effect on this virus.

Inhibition by caffeic acid of the influenza A virus multiplication in vitro

Caffeic acid has been shown to inhibit the multiplication of influenza A virus in vitro, whereas caffeine, quinic acid and chlorogenic acid do not. Caffeic acid has also been shown to have antiviral activity against herpes simplex virus (DNA virus) and polio virus (RNA virus). In the present study, a comparison of the one-step growth curve of the influenza virus in the presence of caffeic acid with that in the absence of the reagent showed that an eclipse period of the virus multiplication in the infected cells was not affected by the reagent, while the progeny virus yield was markedly decreased in the presence of caffeic acid. In additional experiments, it was found that the addition of caffeic acid at an early time point post-infection (within 3 h post-infection) was mandatory for extensive antiviral activity, suggesting that a major target of the reagent exists in the early stages of infection. Simultaneously with the decrease in the progeny virus yield, both the virus-induced cytopathic effects and apoptotic nuclear fragmentation were markedly suppressed by the reagent, suggesting that caffeic acid suppresses, at least temporally, the degeneration of the virus-infected cells and that the observed antiviral activity is likely not the secondary result of the cytotoxic effects of the reagent. These results suggest the potential pharmacological use of caffeic acid or its derivatives as an antiviral drug against influenza A virus.

Antimicrobial Activity and Stability of Weakly Acidified Chlorous Acid Water

The antimicrobial activity of weakly acidified chlorous acid water (WACAW) against Staphylococcus aureus, non-pathogenic Escherichia coli, enterohemorrhagic E. coli (EHEC O157:H7), Candida albicans, and spore-forming Bacillus and Paenibacillus species was evaluated in vitro. The antiviral activity was also examined using feline calicivirus (FCV). Diluted WACAW (>100 ppm) effectively reduced the number of non-spore-forming bacteria (>4 log10 CFU reductions) within 5 min. Treatment with this sanitizer at 400 ppm for 30 min achieved>5 log10 CFU reductions in spore-forming Bacillus and Paenibacillus species while an equivalent concentration of sodium hypochlorite (NaClO) resulted in only a 0.98 and 2.72 log10 CFU reduction, respectively. The effect of this sanitizer against FCV was equivalent to that of NaClO. Immersion in WACAW (400 ppm) achieved >4 and 2.26 log10 CFU reductions in Campylobacter jejuni and EHEC, respectively, on artificially contaminated broiler carcass pieces. Finally, theantimicrobial activity of this sanitizer was shown to be maintained for at least 28 d when in contact with nonwoven fabric (100% cotton). This study showed that pH control of chlorous acid is expected to modify its antimicrobial activity and stability. WACAW is expected to have applications in various settings such as the food processing and healthcare industries.

Arginine inactivates human herpesvirus 2 and inhibits genital herpesvirus infection

Arginine, among the amino acids, has demonstrated unique properties, including suppression of protein-protein interactions and virus inactivation. We investigated the effects of arginine on the infectivity of human herpesvirus 2 (HHV-2) and the potential application of arginine as a chemotherapeutic agent against genital herpes. Arginine directly inactivated HHV-2 and characterization of the inactivation demonstrated that 1 M arginine at pH 4.3 inactivated the virus more efficiently compared to 0.1 M citrate or 1 M sodium chloride, indicating that neither acidic pH nor ionic strength alone is sufficient for virus inactivation. The effect of arginine was rapid and concentration-dependent. Although virus inactivation was efficient at an acidic pH, arginine inactivated the virus even at a neutral pH, provided that a higher arginine concentration and prolonged incubation time were used. In addition, arginine suppressed the multiplication of HHV-2 under the conditions at which its effect on cell viability was insignificant. Pilot mouse model studies revealed a marked suppression of death by arginine when the mice were infected with HHV-2 through the vaginal route, followed by an intermittent application of acidic arginine by vaginal instillation.

Effects of electrolytes on virus inactivation by acidic solutions.

Acidic pH is frequently used to inactivate viruses. We have previously shown that arginine synergizes with low pH in enhancing virus inactivation. Considering a potential application of the acid inactivation of viruses for the prevention and treatment of superficial virus infection at body surfaces and fixtures, herein we have examined the effects of various electrolytes on the acid-induced inactivation of the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), the influenza A virus (IAV) and the poliovirus upon their incubation at 30˚C for 5 min. Eight electrolytes, i.e., phosphate, NaCl, glutamate, aspartate, pyrrolidone carboxylate, citrate, malate and acetate were tested. No detectable inactivation of the poliovirus was observed under the conditions examined, reflecting its acid-resistance. HSV-1 and HSV-2 responded similarly to the acid-treatment and electrolytes. Some electrolytes showed a stronger virus inactivation than others at a given pH and concentration. The effects of the electrolytes were virus-dependent, as IAV responded differently from HSV-1 and HSV-2 to these electrolytes, indicating that certain combinations of the electrolytes and a low pH can exert a more effective virus inactivation than other combinations and that their effects are virus-specific. These results should be useful in designing acidic solvents for the inactivation of viruses at various surfaces.