CD4+ T cells have a crucial role in mediating protection against a variety of pathogens through production of specific cytokines. However, substantial heterogeneity in CD4+ T-cell cytokine responses has limited the ability to define an immune correlate of protection after vaccination. Here, using multiparameter flow cytometry to assess the immune responses after immunization, we show that the degree of protection against Leishmania major infection in mice is predicted by the frequency of CD4+ T cells simultaneously producing interferon-gamma, interleukin-2 and tumor necrosis factor. Notably, multifunctional effector cells generated by all vaccines tested are unique in their capacity to produce high amounts of interferon-gamma. These data show that the quality of a CD4+ T-cell cytokine response can be a crucial determinant in whether a vaccine is protective, and may provide a new and useful prospective immune correlate of protection for vaccines based on T-helper type 1 (TH1) cells.

The HIV-1 Gag polyprotein contains a segment called p2, located between the capsid (CA) and nucleocapsid (NC) domains, that is essential for ordered virus assembly and infectivity. We subcloned, overexpressed, and purified a 156-residue polypeptide that contains the C-terminal capsid subdomain (CA CTD ) through the NC domain of Gag (CA CTD -p2-NC, Gag residues 276-431) for NMR relaxation and sedimentation equilibrium (SE) studies. The CA CTD and NC domains are folded as expected, but residues of the p2 segment, and the adjoining thirteen C-terminal residues of CA CTD and thirteen N-terminal residues of NC, are flexible. Backbone NMR chemical shifts of these 40 residues deviate slightly from random coil values and indicate a small propensity toward an ␣-helical conformation. The presence of a transient coil-tohelix equilibrium may explain the unusual and necessarily slow proteolysis rate of the CA-p2 junction. CA CTD -p2-NC forms dimers and self-associates with an equilibrium constant (K d ‫ס‬ 1.78 ± 0.5 M) similar to that observed for the intact capsid protein (K d ‫ס‬ 2.94 ± 0.8 M), suggesting that Gag self-association is not significantly influence by the P2 domain.Keywords: NMR; protein structure and dynamics; HIV-1; p2The genome of the human immunodeficiency virus type-1 (HIV-1) encodes an ∼55-kDa polyprotein, called Gag, that is itself sufficient for assembly of virus-like particles (Gheysen et al. 1989;Wills and Craven 1991). Several thousand copies of Gag self-associate at lipid rafts on the plasma membrane and bud to form an immature virion. Subsequent to budding, Gag is proteolytically cleaved by the viral protease into the following mature proteins and peptide fragments (listed from N terminus to C terminus): matrix (MA), capsid (CA), p2, nucleocapsid (NC), p1, and p6 (Fig. 1A). Cleavage leads to a dramatic morphological change, termed maturation (Vogt 1996), in which the mature CA proteins assemble into the conical core particle that encapsidates two copies of the viral genome, the NC proteins coat the viral genome, and the MA proteins remain associated with the viral envelope (Coffin et al. 1997).Although the structures and functions of the mature, functional MA, CA, and NC proteins have been well characterized (Turner and Summers Abbreviations: HIV-1, human immunodeficiency virus type-1; CA CTD , capsid C-terminal domain protein; CA p24, full-length capsid protein; p2, 14-residue protein within Gag; NC, nucleocapsid protein; MA, matrix protein; SE, sedimentation equilibrium; K d , equilibrium dissociation constant; NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum correlation; HNCA, triple resonance experiment; HN(CO)CA, triple resonance experiment; 2D, two-dimensional; 3D, three-dimensional; NOESY, nuclear Overhauser effect spectroscopy; R 1 (T 1 ), longitudinal relaxation rate (time); R 2 (T 2 ), transversal relaxation (time); XNOE, heteronuclear 15 N{ 1 H} nuclear Overhauser effect; TFE, 2,2,2-trifluoroethanol; CSI, chemical shift index.Article published online ahead of...

The quality of a Th1 response can be a prospective correlate of vaccine-mediated protection against certain intracellular pathogens. Using two distinct vaccine platforms, we evaluate the influence of interleukin (IL) 10 production on the magnitude, quality, and protective capacity of CD4+ T cell responses in the mouse model of Leishmania major infection. Multiparameter flow cytometry was used to delineate the CD4+ T cell production of interferon (IFN) γ, IL-2, tumor necrosis factor (TNF), and IL-10 (or combinations thereof) after vaccination. Immunization with a high dose of adenovirus (ADV) expressing leishmanial proteins (MML-ADV) elicited a limited proportion of multifunctional IFN-γ+IL-2+TNF+ Th1 cells, a high frequency of IL-10–producing CD4+ T cells, and did not protect against subsequent challenge. Surprisingly, in the absence of IL-10, there was no change in the magnitude, quality, or protective capacity of the Th1 response elicited by high-dose MML-ADV. In contrast, after immunization with MML protein and CpG (MML + CpG), IL-10 limited the production of IL-12 by DCs in vivo, thereby decreasing the generation of multifunctional Th1 cells. Consequently, three immunizations with MML + CpG were required for full protection. However, inhibiting IL-10 at the time of immunization enhanced the magnitude and quality of the Th1 response sufficiently to mediate protection after only a single immunization. Overall, we delineate distinct mechanisms by which vaccines elicit protective Th1 responses and underscore the importance of multifunctional CD4+ T cells.