Natth Bhamarapravati

Dengue 2 PDK-53 Virus as a Chimeric Carrier for Tetravalent Dengue Vaccine Development

ABSTRACT Attenuation markers of the candidate dengue 2 (D2) PDK-53 vaccine virus are encoded by mutations that reside outside of the structural gene region of the genome. We engineered nine dengue virus chimeras containing the premembrane (prM) and envelope (E) genes of wild-type D1 16007, D3 16562, or D4 1036 virus within the genetic backgrounds of wild-type D2 16681 virus and the two genetic variants (PDK53-E and PDK53-V) of the D2 PDK-53 vaccine virus. Expression of the heterologous prM-E genes in the genetic backgrounds of the two D2 PDK-53 variants, but not that of wild-type D2 16681 virus, resulted in chimeric viruses that retained PDK-53 characteristic phenotypic markers of attenuation, including small plaque size and temperature sensitivity in LLC-MK2 cells, limited replication in C6/36 cells, and lack of neurovirulence in newborn ICR mice. Chimeric D2/1, D2/3, and D2/4 viruses replicated efficiently in Vero cells and were immunogenic in AG129 mice. Chimeric D2/1 viruses protected adult AG129 mice against lethal D1 virus challenge. Two tetravalent virus formulations, comprised of either PDK53-E- or PDK53-V-vectored viruses, elicited neutralizing antibody titers in mice against all four dengue serotypes. These antibody titers were similar to the titers elicited by monovalent immunizations, suggesting that viral interference did not occur in recipients of the tetravalent formulations. The results of this study demonstrate that the unique attenuation loci of D2 PDK-53 virus make it an attractive vector for the development of live attenuated flavivirus vaccines.

DENGUE-3 (16562) PGMK 33 VACCINE : NEUROVIRULENCE, VIREMIA AND IMMUNE RESPONSES IN MACACA FASCICULARIS

Investigation of monkey neurovirulence of dengue-3 viruses (DEN-3, 16562) was undertaken to provide an evaluation of the relative safety of virus strain attenuated for potential use of live virus vaccine. Ten flavivirus-negative, cynomolgus monkeys (Macacafascicularis) were used in the test. The animals were inoculated intrathalamically, intraspinally and intramuscularly with DEN-3 PGMK 33 attenuated live virus vaccine (6 monkeys): parent virus (2) and control cell culture fluid (2). Blood samples were collected on days 0, 1, 2, 4, 6, 8, 10, 12 and 21 for virus isolation and days 0 and 21 or 22 for serologic testing. One monkey with DEN-3 (16562) PGMK 33 candidate vaccine had detectable viremia on day 10. By day 21, all recipients of PGMK 33 and both monkeys with DEN-3 parent virus developed serum neutralizing antibodies to DEN-3 titers ranged from 56-320. The monkeys showed no evidence of illness and none died of dengue infection. Histopathological examination of tissue collected on day 21 or 22 revealed only minimal neurovirulence lesions as scored by the routine grading system. No differences were observed between the DEN-3 parent and vaccine viruses and it is concluded that neither virus is neurovirulent for cynomolgus monkeys.

Langerhans cell density and serological changes following intradermal immunisation of mice with dengue 2 virus

After the introduction of the dengue-2 (16681) virus by intradermal (i.d.) injection into the footpads of mice, Langerhans cells (LCs) increased in numbers within 24 h at the site of injection and neutralising antibody developed. On comparing the i.d. and intramuscular (i.m.) routes, antibody was produced more rapidly and at higher levels when the virus was injected by the i.d. route. Subsequent re-challenge by the i.d. route produced an even more rapid serological response with all mice producing significant neutralising titres within 12 h. Numbers of ATPase-positive LCs varied with time. A significant sharp drop in LC densities in the early post-injection phase directly correlated with the increased numbers of dendritic cells in the superficial dermis and interfollicular sinuses of draining lymph nodes (LN). Immunofluorescence showed the presence of viral antigen in the footpad epidermis and draining LN within minutes or within 2 h of challenge, respectively.

Book Review / Announcements

Sydney E. Salmona Clinics in Haematology, vol. 11, No. 1 Myeloma and Related Disorders Saunders, Eastbourne 1982 VII + 238 pp.; E 10.75, bound ISSN 0308-2261 Within the 238 pages of this very legible text the authors have presented a broad, yet concise review of the current concepts of myeloma in the clinic and in the laboratory. The clinician concerned with the treatment of myeloma patients is provided with guidelines in distinguishing between disease categories requiring systemic treatment, the localized and extramedullary plasmacytomas best treated locally and those with monoclonal gammopathy of undetermined significance (MGUS) probably best not treated at all initially. The prognosis and therapeutic results to be expected for these various types of presentation are taken from the authors own experience and cooperative group studies. Insight into the future prospects of treatment are given not only for currently available cytostatic agents, but also for interferon as well as in vitro cloning and chemosensitivity. Such prospects are also hinted at in the chapter on immunoregulato-ry circuits in myeloma. Those seeking fundamental information regarding the pathogenic mechanisms involved in the frequent complications of this disease such as infection and humoral immunoinsufficiency, hypercalcemia, and renal insufficiency will be rewarded. The chapter on amyloidosis depicts this disease in its various forms as to clinical presentation, diagnosis, laboratory findings, and therapeutic possibilities. Last but not least, the chapter on plasma cell neoplasms and acute leukemia places the connection of these two entities in its proper perspective according to present-day knowledge and experience. The possible role of cytostatic treatment in this relationship, either direct or indirect, is discussed in detail. This book provides the student of this disease in any or all of its aspects with enjoyable and informa tive reading. The ample list of references with each chapter should more than satisfy all readers requir ing more detail. R. Sonntag, Bern Buenos Aires, Argentina, September 1-7, 1984 President: Prof. L.J. Bergna