Comparative analysis of HIV-1 recombinant envelope glycoproteins from different culture systems.

The productivity of stable Chinese hamster ovary cell lines secreting HIV-1 monomeric (IIIB gp120) and oligomeric (UG21 gp140) recombinant envelope glycoproteins was compared in serum-containing (S+), serum-free (S-) and protein-free (P-) culture media. UG21 gp140 expression was greatest in S+ medium, while IIIBgp120 production was lower than gp140 in all three media but highest in S-. UG21 gp140 production was highest in standard 850-cm2 roller bottle cultures in S+ media, peaking after 14 days of incubation, while expression levels in the three media were 0.5 (S+), 0.4 (S-) and 0.2 (P-) mg/l, from which 90, 80 and 12% of gp140, respectively, could be purified by immunoaffinity chromatography. Purified UG21 gp140 from S+ and S- media possessed biological functionality as evidenced by CD4 and monoclonal antibody (Mab) binding. In contrast, UG21 gp140 from P- medium appears to be misfolded and non-functional. Despite the possession of a different N-linked glycan profile, UG21 gp140 from S- media shows very similar CD4 and Mab binding characteristics to S+ UG21 gp140. The relevance of these findings to HIV vaccine development is discussed.

Expression and characterisation of recombinant oligomeric envelope glycoproteins derived from primary isolates of HIV-1.

The production, purification and characterisation of recombinant gp140 oligomeric envelope glycoproteins derived from six primary isolates of HIV-1 (covering clades A, B, C, D, F and O) are described. Using a Chinese hamster ovary cell expression system, expression levels of between 0.1 and 1 mg/l cell-conditioned culture media were obtained, and purified to >95% by affinity chromatography. A, B, D, F and O clade gp 140s were found to be multimeric, bind to a panel of defined env-specific monoclonal antibodies and interact with CD4 and CXCR4, demonstrating correct folding. Their immunogenicity was confirmed by the generation of high-titre anti-gp140 antibodies in rabbits. The C clade gp140 was incorrectly folded and poorly antigenic. Despite the presence of an unmodified gp120/41 cleavage site, only the B clade gp140 showed significant processing to gp120 and gp41. Each gp140 has a specific pattern of oligomerisation, and varies in its resistance to reducing agents and salt concentration. The binding of gp140 to soluble and cell-surface CD4 and CXCR4 is related to the degree of oligomerisation. The C1 and C5 regions, CD4 binding domain and the epitope defined by the 2G12 monoclonal antibody were well exposed, but the C-terminal region of the extracellular domain of gp41 appears to be occluded by oligomerisation. These reagents have potential as immunogens for use in vaccine development.

Analysis of available measures for malaria control in Africa south of the Sahara.

Africa south of the Sahara is not homogeneous and presents several extreme conditions where malaria persistence is ensured by a complex and highly adaptable vector system. Plasmodium falciparum is the most widespread and life threatening of the malaria parasites of man, particularly for young children and pregnant women. Large-scale residual spraying was not totally effective and was very costly, and mass chemoprophylaxis was not feasible. The spread of chloroquine resistance added arguments against uncontrolled use of drugs. Chemoprophylaxis is now recommended only for pregnant women, especially in their first pregnancy, whilst chloroquine 25 mg base/kg over 3 days is recommended for curative treatment in villages. Second line treatment regimens should be available, together with the possibility of referring severe malaria cases quickly to appropriate clinical facilities. Other control measures include self-protection against mosquito bites by bednets (especially those impregnated with synthetic pyrethroids), mosquito coils, repellents, window and door screening; other measures to prevent man-mosquito contact, such as careful siting of settlements and zooprophylaxis; anti-larval measures, i.e. source reduction, protection of wells and water reservoirs, larviciding, introduction of larvivorous fish; and sprays against adult mosquitoes. The elaboration of strategies for control and their application requires a study of the existing situation. A core of specialists is required in each country, to help with decentralized planning and evaluation of malaria control and to ensure quality control of services, training and applied field research. Additional measures may become available in the future, especially anti-malaria vaccines, and countries should be ready to study their application.

Recent applied field research activities carried out in tropical Africa.

A review has been undertaken of applied field research in malaria in tropical Africa from 1975 onwards, the aim being to show recent trends and to emphasize the needs for further research in support of malaria control.Studies are grouped according to whether they relate to parasites, vectors, epidemiology, or control. The first group is concerned mainly with the study of the appearance and development of resistance of Plasmodium falciparum to drugs. The second group deals with vector bionomics and the differentiation of various species in the Anopheles gambiae complex. Next come descriptive and analytical surveys and studies on the characterization of malaria as a health and social problem, the importance of some congenital factors, and immunological aspects of the disease. Studies on control comprise the use of drugs, insecticides, and biological methods.The main achievements of research to date have been to improve knowledge of the distribution of chloroquine resistance, which is still mainly confined to East Africa; to clarify the distribution of the components of the A. gambiae complex, even in the formerly known A. gambiae sensu stricto; and to provide indications that the use of mass chemosuppression may favour drug resistance and reduce the malaria antibodies in the population, although the clinical significance of the latter needs to be elucidated.Among the domains considered important for future research are the monitoring of drug sensitivity, not only to 4-aminoquinolines but also to alternative drugs; the determination of optimum drug regimens in various circumstances and population groups; studies on malaria mortality, morbidity, and immunity as related to the use of drugs; the study of the epidemiological importance of various vector species, their behaviour and amenability to control; and feasibility studies on various methods of control in the context of the primary health care settings, including cost-effectiveness and cost-benefit determination.