The assay was performed once in triplicate, and the results are expressed as mean % neutralization values for each rabbit. The neutralizing potential of the antibodies from the best four (±)-BAY-1251152 rabbits (% neutralization>median value) from the S+E2-S and S+E2-S+apoE groups was analyzed by calculating the ratio of % neutralization to the corresponding anti-E2 responses (OD 490nm) at day 56. lowered the incorporation of E2-S protein. Immunization of New Zealand rabbits resulted in similar anti-S responses for all those rabbits, whereas anti-E1/-E2 antibody titers varied according to the presence or absence of apoE. Regarding the neutralizing potential of these anti-E1/-E2 antibodies, it was higher in rabbits immunized with apoE-bearing particles. In conclusion, the association of apoE with HCV envelope proteins may be a great strategy for improving HCV vaccines based on viral envelope proteins. Subject terms:Hepatitis C, Protein vaccines, Hepatitis C virus == Introduction == About 71 million people worldwide are chronically infected with hepatitis C virus (HCV), a major cause of severe chronic liver disease that may progress (±)-BAY-1251152 to hepatocellular carcinoma (HCC)1,2. Therapies based on the use of direct-acting antivirals (DAAs) are available and highly effective (with cure rates above 95%), but with some limitations. Most infected individuals are asymptomatic, so only a small percentage are diagnosed and, therefore, treated. The cost of DAAs, which has decreased in recent years, is less of a problem than the cost of the screening logistics required to identify chronic carriers of HCV worldwide that have yet to be treated3. Moreover, reinfection can RGS1 occur, because these drugs have no protective effect, and they do not fully prevent the development of severe liver diseases, such as HCC, either46. DAAs can also fail to cure infections involving several distantly related genotypes of HCV79. The World Health Organization (WHO) aims to eliminate viral hepatitis as a public health threat by 2030. However, it was estimated in 2015 that two million new HCV infections occur annually, which is much (±)-BAY-1251152 greater than the number of people cured (843,000 individuals)1. Recent studies have suggested that this WHOs goals are likely to be reached only locally in a few countries, not globally10,11. In light of this evidence, it will be difficult to control the spread of HCV without a prophylactic vaccine. The spontaneous clearance of HCV has been observed in 1525% of patients and is related to early cellular and humoral responses1214. This early induction of neutralizing antibodies (Nabs), together with a T-cell response, is the key to containing the infection and provides important leads for the development of a prophylactic vaccine. Nevertheless, a randomized clinical trial phase 1/2 (NCT01436357) resulted in the induction of T cell responses but did not prevent the development of chronic contamination in comparison to the placebo group, which may suggest that B cell responses may be essential in the prevention of the disease progression15. In fact, most of the preventive vaccines against various pathogens currently available are based on the development of Nabs16. In the case of HCV, the envelope glycoproteins (E1 and E2) are the natural targets of these antibodies and have been widely used in various prototype vaccines1721. One of these candidate vaccines, based on recombinant HCV E1 and E2 proteins, was shown to elicit high titers of anti-HCV envelope antibodies with cross-neutralizing properties, together with a T-cell response, in a human phase I clinical trial (NCT00500747)17,22. However, the E1 and E2 glycoproteins are transmembrane proteins and are thus difficult to extract and purify in large-scale manufacturing processes. We have tried to overcome this problem by developing a prototype HCV vaccine consisting of full-length HCV E1 or E2 glycoproteins (from genotype 1a) fused to the heterologous hepatitis B virus (HBV) small envelope protein (S), also known as small hepatitis B surface antigen (SHBsAg), that self-assemble into highly immunogenic, noninfectious and secreted subviral particles (SVPs) resembling the HBV vaccine23. We have shown that these particles can induce strong specific antibody responses directed against the HCV and HBV envelope proteins in immunized rabbits, together with Nabs capable of neutralizing (±)-BAY-1251152 HCV contamination in vitro24,25. Moreover, we have shown that this E1 and E2 proteins are more immunogenic when presented separately in the SVPs26. Finally, we recently reported that immunization with mixtures of SVPs presenting HCV envelopes of different genotypes improves the induction of cross-neutralizing antibodies against various genotypes of HCV27. However, it is important to take the close interactions of HCV with host lipid metabolism into account when developing a vaccine against this virus. HCV associates with lipid components and circulates in the bloodstream of infected patients as lipoviral particles (LVPs), which help the virus (±)-BAY-1251152 to escape from Nabs28. Apolipoprotein E (apoE) is usually a component of HCV LVPs that has been.