A prime-and-trap vaccination strategy to evaluate minimal requirements for malaria liver stage protection in mice

Abstract

Background

The Plasmodium liver stage is an attractive prophylactic vaccine target since blocking parasites in the liver has the potential to prevent both clinical disease and transmission. However, immunity to Plasmodium liver stages is difficult to study, and highly efficacious liver stage vaccines have been difficult to develop. Prime-and-trap vaccination induces liver CD8⁺ T cells that can provide complete liver stage protection in mice. However, the minimal vaccine requirements for complete liver stage protection remain unclear.

Methods

In this study, a prime-and-trap vaccination strategy was used to evaluate minimal vaccine requirements for malaria liver stage protection in mice. Here, Prime-and-trap pulls and traps primed Plasmodium yoelii circumsporozoite protein (CSP)-specific CD8⁺ T cells in the liver via liver expression of DNA-encoded CSP delivered by hydrodynamic transfection (HDT). HDT-CSP trapping doses were progressively de-escalated to define the minimal requirements for inducing CSP-specific CD8⁺ T cells in the liver.

Results

Prime-and-HDT trap induced sterile protection against sporozoite challenge with CSP-HDT doses as low as 5 ng DNA. However, protection was lost at an ultra-low CSP-HDT dose of 50 pg, which was associated with a similarly low level of CSP-specific CD8⁺ T cells in the liver. These results suggest that there is a threshold of CSP-specific CD8⁺ T cells required in the liver to achieve sterile protection.

Conclusions

Overall, these studies indicate that prime-and-HDT trap vaccination offers a tractable new strategy to study immunity to liver stage malaria in mice that is inexpensive, reproducible, and accessible.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12936-026-05940-x.

Background

The Plasmodium liver stage (LS) is an attractive prophylactic vaccine target but is more difficult to study than the blood stage. Blocking parasites in the liver has the potential to prevent both clinical disease and transmission. However, highly efficacious LS vaccines have been difficult to develop, due in part to the inaccessibility of the human liver for immunological analysis. We thus rely on preclinical animal studies to understand LS protective immune responses. Studies in mice have demonstrated that while sporozoite (spz)-neutralizing antibodies contribute to protection, liver CD8⁺ tissue-resident memory T cells (Trm) are critical for complete and durable LS protection [1].

Protective liver CD8⁺ Trm cells can be induced by prime-and-trap vaccination [1–5]. Prime-and-trap is a two-step vaccine strategy designed to induce such protective LS immune responses. In step one, peripheral Plasmodium-specific CD8⁺ T cells are ‘primed’ via systemic vaccination. In step two, the Plasmodium-specific CD8⁺ T cells are pulled and ‘trapped’ in the liver. Effective trapping requires the targeted expression of vaccine antigen in the liver, which provides the localized signals to recruit Plasmodium-specific T cells to the liver. Many different prime-and-trap strategies have been described [1–5], but all share this same fundamental mechanism.

Hydrodynamic transfection (HDT) is a preclinical research method to specifically deliver plasmid DNA (pDNA) to the liver of rodents [6, 7]. It involves rapid intravenous (IV) injection of pDNA-saline solution equal to 7–10% body weight. This generates high intravascular pressure that facilitates the delivery of pDNA directly into liver cells, mostly hepatocytes. In contrast to standard slow infusion IV injection, pDNA-HDT preferentially targets the liver with up to 40% of hepatocytes transfected [6]. Thus, HDT achieves highly efficient and liver-specific transfection, which makes HDT a useful tool to study immunity to the Plasmodium LS [8, 9].

Despite recent advances to studying liver T cells, there are many outstanding questions about requirements to induce protective LS immune responses. The goal of this study was to develop a simple, affordable, and reproducible vaccination strategy to study LS protective immune responses in mice. Specifically, we set out to understand how protective CD8⁺ T cells are formed in the liver after vaccination. pDNA-HDT is a simple and reliable method to achieve specific transgene expression in the liver. Thus, we hypothesized that we could use a prime-and-HDT trap vaccination strategy to define the minimal requirements for liver stage protection.

Methods

Mice: BALB/cJ mice were purchased at 4–6 weeks old (Strain #:000651, Jackson Laboratories, Barr Harbor, ME). Mice were housed at the University of Washington in an Institutional Animal Care and Use Committee-approved animal facility and were used under an approved protocol (4317-01 to S.C.M.). All mice were female unless otherwise specified in the figure legend.

DNA Plasmids: Endotoxin-free pDNA were constructed in the pUb.3 vector as described previously [2, 10] and sequences are listed in Supplementary Fig. 1. Briefly, two versions of Plasmodium yoelii (Py) circumsporozoite protein (CSP) pDNA were utilized: a minigene encoding the SYVPSAEQI epitope (CSP epi) and a full-length version without the QGPGAP and PPQQ major repeat regions (CSP FLNR). Py ribosomal protein L3 (RPL3), RE9h red-shifted luciferase (Luc), and Escherichia coli heat-labile toxin (LT) plasmids were also similarly constructed as previously described [2, 11, 12]. All plasmid stocks were sequenced via Sanger Sequencing before use (GeneWiz Inc.). All Py CSP vaccines utilized the CSP epi plasmid (herein referred to as ‘CSP’) unless otherwise noted in the figure legend.

Gene Gun: Gene gun (gg) pDNA vaccine cartridges were constructed as previously described [2]. Briefly, the vaccine plasmids plus 1:10 LT adjuvant plasmid were loaded onto gold beads (Technic Inc.) to produce gene gun vaccine cartridges (0.5 µg DNA per cartridge). Mice were vaccinated on the abdomen using a PowderJect-style gene gun with two cartridges per vaccination day (1 µg total DNA). Mice were gg primed on Days 0 & 2 and gg boosted on Day 28. This method of prime-boost vaccination with CSP/LT-encoding plasmids via gg is referred to as ggCSP prime-boost (P + B).

Hydrodynamic Transfection (HDT): HDT methods were previously described [2] and adapted from prior studies [13, 14]. Briefly, anesthetized mice were weighed and 10% (v/w) pDNA in saline was injected IV via tail vein in 5–10 s using a 27.5-gauge needle and a 3 mL syringe. The dose of pDNA administered via HDT ranged from 50 pg to 20 µg pDNA, with specific doses described for each experiment. For this study three dose groups were utilized: standard (1.2–20 µg), low (5-200 ng), or ultra-low (50 pg) CSP-HDT doses. To validate the success of the HDT injection, for some experiments 3-5 µg Luc pDNA was mixed with CSP or RPL3 pDNA and co-administered via HDT. HDT details are described in the figures for each experiment.

Sporozoite Challenge: Female Anopheles stephensi mosquitoes were reared and infected with wild-type Py 17XNL (PyWT) at the Seattle Children’s Research Institute Insectary or the University of Washington Insectary (Seattle, WA). 14–18 days later, PyWT sporozoites (spz) were dissected and purified from infected mosquito salivary glands [2, 4]. PyWT spz were counted via hemocytometer and diluted in Schneider’s media (Gibco). For all spz challenge administrations, 1 × 10² freshly-dissected PyWT spz in 100 µL were IV injected retro-orbitally. Naïve ‘infectivity control’ animals were included in all challenge studies to verify the infectivity of PyWT spz. Blood stage parasitemia was assessed by Giemsa (Sigma-Aldrich) stained thin blood smear microscopy on Days 3–14 post-challenge. Mice were deemed protected if blood smears remained negative for parasites up to Day 14.

In Vivo Imaging System (IVIS): Live in vivo imaging of Luc protein expression following Luc pDNA administration via HDT was as previously described [2]. Briefly, bioluminescence was evaluated by intraperitoneal administration of 50 mg/mL D-luciferin (Gold Biotechnology) followed by IVIS. IVIS images were analyzed using Living Image 3.0 software (Perkin-Elmer) with regions of interest (ROI) placed around the liver. All mouse abdomens were shaved within 24 h of IVIS.

Liver leukocyte and splenocyte ELISPOT: Liver leukocytes and splenocytes were isolated as described [4, 10]. Briefly, liver and spleen cells were strained through 100 μm filters, subjected to ammonium-chloride-potassium lysis, and final cell pellets were resuspended in RPMI 1640 supplemented with glutamine and 10% fetal bovine serum. PyCSP (SYVPSAEQI) peptides were synthesized by Genemed Synthesis (San Francisco, CA) and reconstituted in DMSO. Mouse IFNγ ELISPOT (eBioscience, San Diego, CA) was conducted by stimulating cells with peptide (or DMSO control) at 1 µg/mL for ~ 18 h at 37 °C and developed following manufacturer guidelines [10]. The number of spot-forming units (SFU) was determined using an ImmunoSpot 5.1 Analyzer (Cellular Technology Limited), and SFU were normalized to DMSO wells (nSFU).

Statistics: Comparisons of IVIS and ELISPOT groups were done using non-parametric Kruskal–Wallis one-way analysis of variance with Dunn’s multiple comparisons test unless otherwise specified in the figure legend. Protection data was evaluated using Fisher’s exact test. Error bars in all figures are reported as the 95% confidence interval (CI) of the median with individual mouse samples shown if applicable. All p-values and individual experiment statistics are listed in corresponding figure legends. Statistical significance was defined as p < 0.05. Prism GraphPad 9.1.2 Software (San Diego, CA) was used for all calculations.

Results

Prime-and-HDT trap vaccination is protective against PyWT spz challenge. Previously we reported that prime-and-HDT trap vaccination pulled and trapped peripheral CSP-specific CD8⁺ T cells in the liver [2]. We also found that HDT transfection and expression of CSP in the liver could drive cytotoxic liver CD8⁺ Trm cell formation [2]. Here, we built upon our previous studies and determined if prime-and-HDT trap was protective against PyWT spz challenge. Mice were prime-boost (P + B) vaccinated with ggCSP to induce CSP-specific CD8⁺ T cells in the periphery. Five days later, at the peak of the peripheral T cell response, CSP-specific CD8⁺ T cells were pulled and trapped in the liver via HDT (Fig. 1A). We utilized Luc pDNA as a reporter to verify successful HDT injection (Fig. 1B-C) and RPL3 as a known immunogenic but non-protective malaria vaccine antigen [11]. As previously described, luminescence was significantly reduced in in mice previously vaccinated with ggCSP indicating rapid killing of CSP-expressing cells [2]. Mice were challenged with 1 × 10² fresh infectious PyWT spz four weeks later. We found that CSP-HDT induced high levels of sterile protection at the four-week challenge timepoint (Fig. 1D). As expected, RPL3-HDT, Luc-HDT, CSP-HDT only, or ggCSP-only control mice showed little to no protection (Fig. 1D–E). This data suggests that prime-and-HDT trap induces sufficient levels of CSP-specific CD8⁺ T cells to protect mice against spz challenge at a memory time point.

Fig. 1.

Prime-and-HDT trap vaccination is protective against PyWT spz challenge. A Experimental design of Prime-and-HDT trap vaccination studies. B IVIS total flux of mice 24 h post HDT. Error bars represent the 95% CI of the median of a subset of the mice from (A). IVIS data was analyzed with Mann Whitney test, *p < 0.05. C Representative IVIS images of the mice from (B). D–E Results of protection studies after challenge with 1 × 10² purified PyWT spz administered four weeks after HDT trap. Protection data from N = 3–15 mice per group across 1–2 independent experiments. Protection data was analyzed with Fisher Exact Test, **p < 0.01, ns p > 0.05. All prime-boost (P + B) groups were vaccinated with ggCSP (CSP epi) except for the RPL3 group, which was vaccinated with ggRPL3

Ultra-low dose HDT is detectable in the liver for at least 1 month. Next, we investigated whether prime-and-HDT trap could be used to define minimal vaccine requirements for LS malaria protection. The malaria LS is notoriously difficult to study, and many outstanding questions remain about the requirements to induce protective CD8⁺ Trm cells in the liver. Here, we hypothesized that prime-and-HDT trap could be utilized as a model to study minimal requirements to induce liver CD8⁺ T cells. First, we determined the minimum HDT dose required for antigen expression in the liver. We utilized Luc-HDT and IVIS to characterize antigen expression and kinetics in the liver after HDT alone (Fig. 2A). De-escalating amounts of Luc pDNA were administered to naïve mice via HDT (3 µg, 3 ng, 30 pg, or 0). As expected, we found that the liver expression declined with decreasing HDT doses (Fig. 2B and Supplementary Fig. 2). Additionally, the total flux in the liver declined incrementally after HDT over a 1-month period. This data suggested that ultra-low doses of HDT delivered antigen are expressed in the liver and that the model could be used to fine tune minimal requirements for LS protection.

Fig. 2.

Ultra-low dose HDT is detectable in the liver for at least 1 month. A Experimental design of Luc HDT studies. B Compiled IVIS total flux of mice post HDT measured over a 1-month period. Error bars represent the 95% CI of the median of the mice from (A). IVIS data from N = 6–9 mice per group across two independent experiments (N = 3 from one experiment for week 3 and week 4 groups). IVIS data was analyzed with Mixed-Effects analysis with Dunnett multiple comparisons (All groups vs naive), **p < 0.01, ns p > 0.05

Low dose prime-and-HDT trap is protective against PyWT spz challenge. Next, we explored if the trapping dose of CSP-HDT could be reduced while still retaining protection. To investigate this possibility, mice were prime-and-HDT trap vaccinated and challenged as before, with the only modification being the progressive dose de-escalation of CSP-HDT from 2 µg to 5 ng (Fig. 3A). As before, we utilized Luc pDNA as a reporter to verify successful HDT injection (Fig. 3B-C). In our first set of challenge experiments, we found that all doses of CSP-HDT induced high levels of sterile protection at the four-week challenge timepoint (Fig. 3D). As expected, RPL3-HDT or Luc-HDT control mice showed little to no protection (Fig. 3D). Interestingly, similarly vaccinated male mice only showed ~ 40% protection against PyWT spz challenge in all CSP-HDT doses tested (Supplementary Fig. 3). This finding was not unexpected since we recently reported significant sex differences in BALB/cJ mice following prime-and-trap vaccination [15].

Fig. 3.

Low dose Prime-and-HDT trap vaccination is protective against PyWT spz challenge. A Experimental design of low dose Prime-and-HDT trap vaccination studies. B IVIS total flux of mice 24 h post HDT. Error bars represent the 95% CI of the median of a subset of the mice from (A). IVIS data was analyzed with Kruskal–Wallis test with Dunn’s multiple comparisons, ns p > 0.05. C Representative IVIS images of the mice from (B). D–E Results of protection studies after challenge with 1 × 10² purified PyWT spz administered four weeks after HDT trap with Luc (C) or without Luc (D). Protection data from N = 10–15 mice per group across two independent experiments. Protection data was analyzed with Fisher Exact Test, ****p < 0.0001, **p < 0.01, ns p > 0.05. All prime-boost (P + B) groups were vaccinated with ggCSP (CSP epi) except for the RPL3 group, which was vaccinated with ggRPL3

All experiments thus far used the well-characterized and immunogenic CSP epitope pDNA for vaccination to specifically induce CD8⁺ T cell responses. However, as this plasmid only contains a single epitope, cellular responses are more focused than after native antigen exposure. We utilized a CSP FLNR (full-length no repeat) plasmid to further diversify the antigenic landscape, while still limiting the induction of anti-CSP spz neutralizing antibodies and biasing responses toward CD8⁺ T cells. Mice were vaccinated using the same strategy as previously described, but all vaccinations used CSP FLNR pDNA. Both CSP epi and CSP FLNR achieved comparable high levels of protection at the four-week challenge interval (Supplementary Fig. 4).

Finally, we evaluated whether the 3 µg Luc pDNA included in the protection studies as a reporter control could impact the protection outcomes. We repeated the challenge study in female mice without Luc pDNA and further reduced the dose to 50 pg. We found in the absence of Luc, standard (12.5 µg) and low (5 ng) doses of CSP-HDT still induced high levels of sterile protection (Fig. 3E), suggesting Luc does not impact protection. Furthermore, we found protection was significantly reduced at an ultra-low dose of 50 pg CSP-HDT (Fig. 3E). Taken together, the data suggests that low dose prime-and-HDT trap vaccination protects female mice against spz challenge at a memory time point.

Low dose prime-and-HDT trap induces CSP-specific CD8⁺ T cells. Dose de-escalation of CSP pDNA identified a HDT trapping dose of > 50 pg was required for sterile protection against PyWT spz challenge. Since protection was lost between CSP-HDT doses of 5 ng and 50 pg, we investigated whether there would similarly be a difference in the numbers of CSP-specific CD8⁺ T cells. Mice were prime-and-HDT trap vaccinated as before with standard (1.2 µg), low (5 ng), or ultra-low (50 pg) CSP-HDT doses. Luc pDNA was not included in these studies. At four-weeks post HDT, livers and spleens were harvested for CSP-specific IFNγ ELISPOT (Fig. 4A). We found significantly more CSP-specific CD8⁺ T cells in the livers of mice receiving low (median nSFU: 2983, 95% CI: 1190–3938) vs ultra-low (median nSFU: 949, 95% CI: 363–1203) CSP-HDT doses (Fig. 4B). This suggests that the minimum number of CSP-specific CD8⁺ T cells required to achieve protection in our model is > 1000 nSFU per liver. Taken together, we found that in our model there is a threshold of CSP-specific CD8⁺ T cells required for protection that can be induced by CSP-HDT doses between 5 ng and 50 pg.

Fig. 4.

Low dose Prime-and-HDT trap vaccination induces CSP-specific CD8. ⁺ T cells in the liver. A Experimental design of low dose Prime-and-HDT trap vaccination studies. B IFNγ ELISPOT from mice from (A) with liver leukocytes (left) or splenocytes (right) stimulated with CSP peptide (SYVPSAEQI) or DMSO vehicle control. Data was normalized to vehicle control. Error bars represent 95% CI of the median from N = 5–10 mice per group across two experiments (N = 3–5 from one experiment for 1.2 µg CSP liver group and all spleen groups). Data was analyzed with Kruskal–Wallis test with Dunn’s multiple comparisons, ****p < 0.0001, *p < 0.05, ns p > 0.05. All prime-boost (P + B) groups were vaccinated with ggCSP (CSP epi)

Discussion

In this study, we further characterized the prime-and-HDT trap vaccination strategy. Previously, we showed that pre-existing CSP-specific CD8⁺ T cells can be pulled and trapped in the liver by CSP-HDT [2]. Here, we investigated whether prime-and-HDT trap could be used to define the minimal vaccine requirements for LS malaria protection. We demonstrated that prime-and-HDT trap vaccination induces sterile protection against PyWT spz challenge with CSP-HDT doses as low as 5 ng. Protection was lost at an ultra-low CSP-HDT dose of 50 pg, which was associated with a similarly low level of CSP-specific CD8⁺T cells in the liver. Thus, in our model we found that there is a threshold of CSP-specific CD8⁺ T cells required in the liver to achieve sterile protection.

Liver CD8⁺ Trm cells are generally considered sufficient for protection from spz challenge in mice [1]. However, it is not clear what the vaccine requirements are to induce protective levels of liver CD8⁺ Trm cells. Here, we used prime-and-HDT trap vaccination to investigate requirements for protective CD8⁺ T cell formation in the liver. Previous data using the same vaccine strategy demonstrated that ggCSP prime-boost induces CSP-specific CD8⁺ T cells that account for only ~ 4% of blood T cells at four-weeks post vaccination [2]. Keeping this element of the vaccine strategy constant, we varied the trapping dose to evaluate the minimum trapping antigen dose required to recruit these CD8⁺ T cells to the liver and provide LS protection. In this study, we found that low doses (5 ng) of CSP-HDT sufficiently pulled and trapped protective numbers of ggCSP-primed CD8⁺ T cells in the liver. This finding suggests that high levels of CSP-antigen are not required for protection if the CSP-antigen is liver-localized. Thus, in our model, we hypothesize the location of the ‘trapping’ antigen specifically in the liver is likely critical.

Since we utilized CSP epi pDNA for most of these experiments, we infer the observed protection was CD8⁺ T cell mediated. We did not analyze CD8⁺ T cell surface markers and thus we cannot definitively conclude that liver CSP-specific CD8⁺ T cells we measured via ELISPOT are Trm cells. However, using the exact same vaccination strategy, we previously used flow cytometry to show that prime-and-HDT trap vaccination induced CSP-specific CD8⁺ Trm cells in the liver [2]. This finding and the high levels of protection we observed therefore strongly suggest that the liver CSP-specific CD8⁺ T cells measured here are likely Trm cells.

Previous studies demonstrated that up to 40% of liver cells are transfected by pDNA-HDT [6, 16, 17]. Although transgene expression is not exclusive to the liver, prior work found that the transgene expression in the liver post HDT is over a thousand-fold higher than other organs [6]. This means the CSP-HDT trap utilized in our studies is highly specific to the liver. In addition, although HDT does result in a transient spike of ALT in serum, indicative of minor liver damage, the procedure is well-tolerated and results in no other noted toxicity [6]. Thus, pDNA-HDT is an effective method to introduce transgenes specifically in the liver, which makes it a unique tool to study LS immune responses in mice.

Protective immune responses to Plasmodium LS parasites are challenging to study. Mouse models are primarily used to study LS protection from spz challenge due to the ability to sample mouse liver for immunology endpoints [18–20]. However, there is no standard vaccine model that is utilized to study LS immunology, which complicates the interpretation of results and comparisons between studies. Additionally, although many innovative vaccine platforms are in development (e.g., mRNA, self-assembling nanoparticles, live-attenuated sporozoites), many of these technologies are not widely available and/or require specialized manufacturing. Furthermore, even when similar vaccines are utilized, many outstanding variables still must also be considered when interpreting results (e.g., mouse strain/sex, parasite strain, vaccine schedule, dose, administration route, and challenge dose/route). For these reasons, comparing and reproducing LS studies across research groups is difficult. In contrast, prime-and-HDT trap vaccination is an accessible and reproducible vaccination strategy, requiring only pDNA – which is affordable and simple to manufacture. Previous work determined that priming is versatile and effective with numerous DNA (or RNA) priming regimens and administration methods (including gg, electroporation, and/or nanoparticles) [3, 21]. pDNA-HDT trapping is also attractive since it is simple, affordable, and requires no specialized equipment. Overall, this suggests prime-and-HDT trap vaccination could be a useful new strategy for LS protection studies.

One limitation is that prime-and-HDT trap vaccination cannot be translated as-is to the clinic. However, this vaccination strategy is a preclinical tool that can be used to evaluate LS protection in mice using challenge and immunological endpoints. We do not anticipate the translation of this strategy to larger animal models or humans because of the volumetric limits imposed by HDT. Another limitation is that we only utilized a single antigen for protection. Additional studies could investigate protection with other malaria vaccine antigens or combinations of multi-antigen cocktails.

Previous mouse data found that both CSP [2] and RPL6 [22] antigens induce high levels of CD8⁺ T cells and complete LS protection in mice. However, despite inducing high levels of CD8⁺ T cells, the similar ribosomal protein antigen RPL3 achieves no LS protection [11], suggesting a difference in the function or quality of the CD8⁺ T cells induced by RPL3 vs RPL6. This may be dependent on many factors, including the timing of antigen expression, location, and/or abundance. This is an outstanding question about LS immune responses that could be investigated using the prime-and-HDT trap vaccination strategy.

Conclusions

In summary, here we showed that prime-and-HDT trap vaccination is a potentially useful model to study immunity to LS malaria in mice. CSP-HDT can pull and trap malaria-specific CD8⁺ T cells in the liver. These cells were functionally cytotoxic [2] and were sufficient to protect against spz challenge. Prime-and-HDT trap vaccination is thus an attractive new strategy to study immunity to LS malaria in mice as it is affordable, reproducible, and accessible.

Abbreviations

LS

Liver stage

Spz

Sporozoite

Trm

Tissue-resident memory T cell

HDT

Hydrodynamic transfection

pDNA

Plasmid DNA

IV

Intravenous

Py

Plasmodium yoelii

CSP

Circumsporozoite protein

CSP epi

CSP epitope

CSP FLNR

CSP full-length no repeats

RPL3

Ribosomal protein L3

Luc

Luciferase

LT

Escherichia coli Heat-labile toxin

gg

Gene gun

P + B

Prime-boost

PyWT

Wild-type Plasmodium yoelii 17XNL

IVIS

In vivo imaging system

ROI

Regions of interest

SFU

Spot-forming units

nSFU

Normalized spot-forming units

CI

95% Confidence interval

RPL6

Ribosomal protein L6

Author contributions

Conceptualization: FNW, RAR, MJS, SCM Funding acquisition: SCM Investigation: FNW, RAR, ACK, MJS Methodology: FNW, RAR, ACK, MJS Project administration: MJS, SCM Supervision: MJS, SCM Writing – original draft: FNW, RAR, ACK, MJS, SCM Writing – review & editing: FNW, RAR, ACK, MJS, SCM.

Funding

This work was partially funded by NIH/NIAID (U01AI155313 and 1R01AI141857 to S. C. M.). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Data availability

All relevant data generated, analyzed and presented in this manuscript are available on request from the corresponding author.

Declarations

Ethics approval and consent to participate

All mice were used under an approved IACUC protocol (4317–01 to SCM) and in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable.

Competing interests

S. C. M. filed a patent application on selected aspects of the prime-and-trap concept through the University of Washington. S. C. M. has equity in a startup company (Sound Vaccines, Inc.) that is negotiating with the University of Washington for rights to this intellectual property. The relationship between the authors and Sound Vaccines, Inc., has been reviewed by the University of Washington and complies with all University and State of Washington policies on such activities. The other authors have no financial conflicts of interest.