Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r 2 ‫؍‬ 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD 50 ) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.

SummaryThe present study was designed to test the effect of bacterial endotoxin on penetration of viruses into the central nervous system (CNS). As a model we used two neurovirulent viruses that lack neuroinvasive capacity: West Nile virus-25 (WN-25) and neuroadapted Sindbis virus (SVN). Administration of lipopolysaccharide (LPS, 100/zg/mouse) to CD-1 mice, followed by WN-25 inoculation resulted in 83% encephalitis and death, compared with <5% in controls. The results in SVN-inoculated CD-1 mice were quite similar. LPS-treated mice suffered 62% mortality compared with 6% in the nontreated group. No changes in viral neuroinvasiveness were demonstrated in viruses isolated from brains of encephalitic mice, suggesting that neuroinvasion is not due to a selection process for an invasive variant, but to direct penetration of the viruses through the blood-brain barrier (BBB). LPS did not induce WN-25 encephalitis in LPS-insensitive C3H/HeJ mice, compared with 100% neuroinvasion in C3H/HeB mice. Induction of neuroinvasion could be transferred to C3H/HeJ mice by transfusion with serum obtained from LPS-treated, LPSresponsive mice. Passive immunization of CD-1 mice with anti-mTNF antibodies before LPS administration did not prevent LPS-induced WN-25 encephalitis. Furthermore, neutralization of tumor necrosis factor activity in the serum of LPS-treated mice did not abolish its activity, and transfusion-associated encephalitis was observed after the administration of the neutralized serum with WN-25.We suggest that LPS can contribute to virus penetration from the blood into the CNS, a process which turns a mild viral infection into a severe lethal encephalitis. This effect is mediated by soluble factors, and is probably achieved by injury to cerebral microvascular endothelium and modulation of BBB permeability. N'euroinvasiveness (ability to penetrate the central nervous system [CNS] 1) and neurovirulence (ability to establish a lethal infection within the CNS) are the crucial elements that determine the capacity of a virus to cause encephalitis (1, 2). The major routes for virus spread into the brain are through nerves or via the bloodstream. Arboviruses, which are the most common known cause of encephalitis worldwide (3, 4), usually spread hematogenously. The blood-brain barrier (BBB), ultrastructurally localized at the cerebral microvascular endothelium, presents a major obstacle to neuroinvasion from the blood (1, 2, 5).The mode and specificity of viral entry through the BBB into the CNS remains obscure. Factors other than inherent l Abbreviations used in this paper: BBB, blood-brain barrier; CNS, central nervous system; DS, donors' serum; SVN, neuroadapted Sindbis virus; genetic viral traits (1, 6-8) can influence neuroinvasion and allow entrance of noninvasive viruses into the CNS. Among such factors are modulation of BBB permeability by hyperosomotic agents, hypercarbia, hypothermia, and mechanical breach (9-12).Modulation of BBB permeability was also found in experimental bacterial meningitis. Functional and ultras...

This study reports a highly efficient, rapid one-step purification process for the production of the recombinant vesicular stomatitis virus-based vaccine, rVSV-∆G-spike (rVSV-S), recently developed by the Israel Institute for Biological Research (IIBR) for the prevention of COVID-19. Several purification strategies are evaluated using a variety of chromatography methods, including membrane adsorbers and packed-bed ion-exchange chromatography. Cell harvest is initially treated with endonuclease, clarified, and further concentrated by ultrafiltration before chromatography purification. The use of anion-exchange chromatography in all forms results in strong binding of the virus to the media, necessitating a high salt concentration for elution. The large virus and spike protein binds very strongly to the high surface area of the membrane adsorbents, resulting in poor virus recovery (<15%), while the use of packed-bed chromatography, where the surface area is smaller, achieves better recovery (up to 33%). Finally, a highly efficient chromatography purification process with CaptoTM Core 700 resin, which does not require binding and the elution of the virus, is described. rVSV-S cannot enter the inner pores of the resin and is collected in the flow-through eluent. Purification of the rVSV-S virus with CaptoTM Core 700 resulted in viral infectivity above 85% for this step, with the efficient removal of host cell proteins, consistent with regulatory requirements. Similar results were obtained without an initial ultrafiltration step.

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The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) increases the need for a rapid development of efficient vaccines. Among other vaccines in clinical trials, a recombinant VSV-∆G-spike vaccine was developed by the Israel Institute for Biological Research (IIBR) and is being evaluated. The development of an efficient downstream purification process (DSP) enables the vaccine to be advanced to clinical trials. The DSP must eliminate impurities, either process- or product-related, to yield a sufficient product with high purity, potency and quality. To acquire critical information on process restrictions and qualities, the application of in-line monitoring is vital and should significantly impact the process yield, product quality and economy of the entire process. Here, we describe an in-line monitoring technique that was applied in the DSP of the VSV-∆G-spike vaccine. The technique is based on determining the concentrations of metabolites, nutrients and a host cell protein using the automatic chemistry analyzer, Cobas Integra 400 Plus. The analysis revealed critical information on process parameters and significantly impacted purification processes. The technique is rapid, easy and efficient. Adopting this technique during the purification process improves the process yield and the product quality and enhances the economy of the entire downstream process for biotechnology and bio pharmaceutical products.