EVOLUTION: Of Mice . . .

Developing efficient and safe adjuvants for use in human vaccines remains both a challenge and a necessity. Past approaches have been largely empirical and generally used a single type of adjuvant, such as aluminium salts or emulsions. However, new vaccine targets often require the induction of well-defined cell-mediated responses in addition to antibodies, and thus new immunostimulants are required. Recent advances in basic immunology have elucidated how early innate immune signals can shape subsequent adaptive responses and this, coupled with improvements in biochemical techniques, has led to the design and development of more specific and focused adjuvants. In this Review, I discuss the research that has made it possible for vaccinologists to now be able to choose between a large panel of adjuvants, which potentially can act synergistically, and combine them in formulations that are specifically adapted to each target and to the relevant correlate(s) of protection.

The perfect mix: recent progress in adjuvant research

Oil-in-water emulsions are potent human adjuvants used for effective pandemic influenza vaccines; however, their mechanism of action is still unknown. By combining microarray and immunofluorescence analysis, we monitored the effects of the adjuvants MF59 oil-in-water emulsion, CpG, and alum in the mouse muscle. MF59 induced a time-dependent change in the expression of 891 genes, whereas CpG and alum regulated 387 and 312 genes, respectively. All adjuvants modulated a common set of 168 genes and promoted antigen-presenting cell recruitment. MF59 was the stronger inducer of cytokines, cytokine receptors, adhesion molecules involved in leukocyte migration, and antigen-presentation genes. In addition, MF59 triggered a more rapid influx of CD11b+ blood cells compared with other adjuvants. The early biomarkers selected by microarray, JunB and Ptx3, were used to identify skeletal muscle as a direct target of MF59. We propose that oil-in-water emulsions are the most efficient human vaccine adjuvants, because they induce an early and strong immunocompetent environment at the injection site by targeting muscle cells.

https://www.pnas.org/content/pnas/105/30/10501.full.pdf

Molecular and cellular signatures of human vaccine adjuvants

The path to a successful vaccine adjuvant--'the long and winding road'.

The development of vaccine adjuvants for human use has been one of the slowest processes in the history of medicine. For almost one century, aluminium hydroxide (alum) has been the only vaccine adjuvant approved worldwide. Only in the past decade have two oil-in-water emulsions and one TLR agonist been approved by the European authorities as new vaccine adjuvants. Despite the fact that alum has been injected into billions of people, its mechanism of action is not fully understood. Recently, several reports have greatly increased our knowledge of the molecular and cellular events triggered by alum; however, the contribution of each of these processes to alum adjuvanticity is still unclear. A study published in this issue of the European Journal of Immunology, together with two recent publications, have demonstrated that the NOD-like receptor, pyrin domain containing 3 (Nlrp3)-inflammasome is the molecular target of alum immunostimulatory activity in vitro. Surprisingly, these three studies reported conflicting results on the requirement of the Nlrp3 inflammasome complex for alum adjuvant effects in vivo. This commentary attempts to resolve some of these discrepancies.



See accompanying article http://dx.doi.org/10.1002/eji.200838549

https://onlinelibrary.wiley.com/doi/pdf/10.1002/eji.200838648

Alum adjuvanticity: Unraveling a century old mystery

Although approximately 3 % of the world's population is infected with Hepatitis C virus (HCV), there is no prophylactic vaccine available. This study reports the design, cloning and purification of a single polyprotein comprising the HCV core protein and non-structural proteins NS3, NS4a, NS4b, NS5a and NS5b. The immunogenicity of this polyprotein, which was formulated in alum, oil-in-water emulsion MF59 or poly(dl-lactide co-glycolide) in the presence or absence of CpG adjuvant, was then determined in a murine model for induction of B- and T-cell responses. The addition of adjuvants or a delivery system to the HCV polyprotein enhanced serum antibody and T-cell proliferative responses, as well as IFN-gamma responses, by CD4+ T cells. The antibody responses were mainly against the NS3 and NS5 components of the polyprotein and relatively poor responses were elicited against NS4 and the core components. IFN-gamma responses, however, were induced against all of the individual components of the polyprotein. These data suggest that the HCV polyprotein delivered with adjuvants induces broad B- and T-cell responses and could be a vaccine candidate against HCV.

Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses.

Broad, multispecific CD4(+) and CD8(+) T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of these responses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins in various prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8(+) T-cell responses but low CD4(+) T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4(+) T helper responses but no CD8(+) T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4(+) T helper responses but no CD8(+) T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4(+) and CD8(+) T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.

Induction of Broad CD4+ and CD8+ T-Cell Responses and Cross- Neutralizing Antibodies against Hepatitis C Virus by Vaccination with Th1-Adjuvanted Polypeptides Followed by Defective Alphaviral Particles Expressing Envelope Glycoproteins gpE1 and gpE2 and Nonstructural Proteins 3, 4, and 5

The invention provides HCV fusion polypeptides including truncated or full- length HCV NS5 polypeptides, and a portion of the HCV NS2 polypeptide, fused to at least one other HCV epitope derived from another region of the HCV polyprotein. The fusions can be used in methods of stimulating an immune response to HCV, for example a cellular immune response to HCV, such as activating hepatitis C virus (HCV)-specific T cells, including CD4+ and CD8+ T cells. The method can be used in model systems to develop HCV-specific immunogenic compositions, as well as to immunize against HCV.

Hcv fusion polypeptides

HCV E1E2 MF59-adjuvanted protein plus HCV E1E2 encoding alphavirus vector for eliciting HCV-specific T cells.

The induction of broad CD4+ and CD8+ T cell responses and cross-neutralizing antibodies against the hepatitis C virus (HCV) by vaccinating with Th1-adjuvanted polypeptides followed by defective alphaviral particles expressing envelope glycoproteins gpE1/gpE2 and nonstructural proteins 3, 4 & 5 Running title: Induction of broad anti-HCV responses by an optimized regimen

Yin-Ling Lin

Papers

Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses.

Induction of Broad CD4+ and CD8+ T-Cell Responses and Cross- Neutralizing Antibodies against Hepatitis C Virus by Vaccination with Th1-Adjuvanted Polypeptides Followed by Defective Alphaviral Particles Expressing Envelope Glycoproteins gpE1 and gpE2 and Nonstructural Proteins 3, 4, and 5

Hcv fusion polypeptides

HCV E1E2 MF59-adjuvanted protein plus HCV E1E2 encoding alphavirus vector for eliciting HCV-specific T cells.