Journal: NPJ Vaccines

Article Title: A combination of TLR-4 agonist and saponin adjuvants increases antibody diversity and protective efficacy of a recombinant West Nile Virus antigen

doi: 10.1038/s41541-018-0077-1

Figure Lengend Snippet: Identification and optimization of adjuvant components results in increased neutralizing antibody titers following a single immunization with WN-80E. In order to identify and optimize an SLA containing adjuvant formulation for WN-80E, we have examined the ability of different SLA containing adjuvant formulations to augment neutralizing antibody titers in combination with recombinant WN-80E protein. WN-80E was combined with SLA formulated in Alum (SLA-Alum), in a stable oil-in-water emulsion (SLA-SE), in a liposome (SLA-Lipo) and in liposomes containing QS21 (SLA-LSQ) a . All adjuvants contained 5 µg of SLA. Given elevated titers observed with SLA-LSQ, amounts of QS21 b and SLA c or both in combination (SLA-LSQ) d were optimized in liposomal adjuvants to maximize WNV neutralizing antibody responses, as measured by PRNT. Neutralizing antibodies were assessed 21 days post-immunization ( n = 5/group, a – c , n = 10/group d ). Bars indicate mean PRNT 90 titers, dashed lines indicate the limit of detection of the assay. A dose-dependent increase in PRNT titer was observed following immunization with both QS21-Liposome and SLA-Liposome adjuvants. Significant increases relative to those observed in animals immunized with WN-80E alone are indicated (**** p < 0.0001, *** p < 0.0005, ** p < 0.01, * p < 0.05, one-way ANOVA). A liposomal adjuvant formulation containing 2 µg of QS21 and varied doses of SLA (SLA-LSQ) showed increased PRNT titers relative to WN-80E alone, and the inclusion of SLA resulted in a statistically significant increase in PRNT titer relative to animals immunized with WN-80E + QS21-Liposomes ( # p < 0.01, % p < 0.05, one-way ANOVA)

Article Snippet: QS21 was obtained through in-house purification of Quil A (Brenntag Biosector), or purchased from Desert King (San Diego, CA).

Techniques: Recombinant

Journal: NPJ Vaccines

Article Title: A combination of TLR-4 agonist and saponin adjuvants increases antibody diversity and protective efficacy of a recombinant West Nile Virus antigen

doi: 10.1038/s41541-018-0077-1

Figure Lengend Snippet: Induction of germinal center B cells and Th1 CD4+ T cells following immunization with WN-80E in combination with liposomal adjuvants. The ability of SLA-LSQ liposomal adjuvants to stimulate germinal center (GC) reactions a – c and CD4+ T-cell responses d – h 7 days following a prime immunization with 1 µg WN-80E ( n = 5 mice/group) was investigated. Cytokine expression levels were determined for several characteristic Th1 cytokines including IFNγ (I), TNFα (T), and IL-2 (2). Inclusion of QS21 b or SLA and QS21 c induced significant increases in germinal center B cells in draining lymph nodes. Similarly, immunization with QS21-Liposome adjuvants induced low levels of IFNγ+ CD4+ T cells e , and very few Th1 CD4+ T cells (IFNγ+ IL-2+ TNFα+) only at high QS21 doses g . Addition of SLA-LSQ adjuvants (containing both QS21 and SLA) resulted in an increased number of IFNγ+ cells f , and many of these showed a canonical Th1 phenotype h . Significant increases relative to those observed in animals immunized with WN-80E alone (*) or WN-80E + QS21-Liposomes (^) are indicated (****/^^^^ p < 0.0001, ***/^^^ p < 0.0005, **/^^ p < 0.01, */^ p < 0.05, one-way ANOVA)

Article Snippet: QS21 was obtained through in-house purification of Quil A (Brenntag Biosector), or purchased from Desert King (San Diego, CA).

Techniques: Expressing

Journal: NPJ Vaccines

Article Title: A combination of TLR-4 agonist and saponin adjuvants increases antibody diversity and protective efficacy of a recombinant West Nile Virus antigen

doi: 10.1038/s41541-018-0077-1

Figure Lengend Snippet: Optimized SLA-LSQ adjuvant formulations induce persistent functional immunity in mice. Following a single immunization of WN-80E (1 µg) with or without SLA-LSQ adjuvant (5 µg SLA/2 µg QS21), we investigated the longevity and functionality of antiviral antibody responses. Seven days post-immunization, and consistent with previous findings, we observe an adjuvant-dependent stimulation of CD4+ T cells a and germinal center B cells b . In addition, LSQ induced an increase in the number of WN-80E specific IgG+ antigen-secreting cells (ASC) in the bone marrow 21 days post-immunization. Consistent with induction of a Th1 CD4+ T-cell response, we find a significant increase in the number of IgG2c+ ASC at this timepoint c . Serum was periodically collected from immunized animals for up to 300 days. LSQ adjuvant stimulated a potent virus neutralizing response that peaked 63 days post-injection, and persisted for up to 300 days d . Three hundred days post-injection, all animals were challenged with 10 5 PFU of WNV (NY385-99 strain). Animals immunized with SLA-LSQ adjuvanted vaccine showed 100% survival, while those immunized with WN-80E alone showed survival similar to naïve controls e . Bars in a represent mean percentages of cytokine-positive cells ( n = 5/group), and error bars reflect standard deviation of the mean. Statistical significance was determined against WN-80E immunized animals by one-way ANOVA (* p < 0.05, ** p < 0.005, *** p < 0.0005,**** p < 0.0001)

Article Snippet: QS21 was obtained through in-house purification of Quil A (Brenntag Biosector), or purchased from Desert King (San Diego, CA).

Techniques: Functional Assay, Injection, Standard Deviation

Journal: NPJ Vaccines

Article Title: A combination of TLR-4 agonist and saponin adjuvants increases antibody diversity and protective efficacy of a recombinant West Nile Virus antigen

doi: 10.1038/s41541-018-0077-1

Figure Lengend Snippet: Optimized LSQ adjuvant formulations reduce viral load in hamsters following a single immunization. Syrian golden hamsters ( Mesocricetus auratus , 4−5 weeks old, n = 10/group) were immunized with a low dose of WN-80E (100 ng) with or without liposomal adjuvants containing QS21 or SLA. Twenty-one days following a prime immunization, n = 5 animals were challenged with 10 5 PFU of WNV via the intraperitoneal route. Animals were monitored for survival a for 14 days post-challenge. Serum was collected from all animals on alternate days from days 1−7 post-challenge. At 3 days post-challenge, which represents the peak of WNV replication, animals immunized with WN-80E + SLA-LSQ had no detectable virus titer, in contrast to other adjuvant formulations b . Examination of viral replication kinetics show that animals immunized with WN-80E + SLA-LSQ show no detectable titer at any timepoint c , d . Either QS21 or SLA alone failed to reduce titer relative to antigen alone c . Furthermore, at the reduced antigen doses tested here, SLA-LSQ was able to enhance protection relative to Alum d . Error bars c , d represent standard deviation from the mean. Prechallenge/post-immunization antigen binding e , and virus neutralizing f , antibody titers were measured 21 days post-immunization. Immunization with SLA-LSQ resulted in increased IgG endpoint titers, as well as significant increases in PRNT titers. (* p < 0.05, ** p < 0.005*** p < 0.0005, **** p < 0.0001, one-way ANOVA)

Article Snippet: QS21 was obtained through in-house purification of Quil A (Brenntag Biosector), or purchased from Desert King (San Diego, CA).

Techniques: Standard Deviation, Binding Assay