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Fractionating a COVID-19 Ad5-vectored vaccine improves virus-specific immunity

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Adenoviruses are widely used in vaccine research due to their high insert capacity and robust immunogenicity (1). In particular, adenovirus serotype 5 (Ad5) is among the most immunogenic vaccine vectors available, but over the last decade its clinical use has been hampered by its high seroprevalence in the human population (24). Recently, the COVID-19 pandemic has re-invigorated the clinical use of this widely characterized vector as a vaccine platform for preventing COVID-19.
Vaccines based on Ad5, such as the CanSino vaccine and the Sputnik V vaccine, as well as vaccines based on adenovirus serotype 26 (Ad26) and chimpanzee adenovirus (ChAdOx1), have been used in millions of people worldwide and have shown potent protection against severe COVID-19 disease (57). However, there are current attempts to enhance the overall magnitude and durability of immune responses, for example by altering vaccine dosing and boosting regimens. Optimization of prime-boost regimens may obviate the need for “third boosters,” which are now recommended for people over 65 years old, as well as patients at high risk of severe illness, including immunosuppressed individuals (8, 9). Importantly, the waning of immunity several months post-vaccination is associated with increase in the number of breakthrough infections (10), leading experts to consider additional boosters for the general population.

Before licensure, the clinical testing of vaccine candidates consists of three phases aimed at evaluating safety, immunogenicity and efficacy. In particular, phase I typically involves dose-escalation studies comparing a range of vaccine doses in people who receive the same dose of the vaccine during the prime and the boost. However, vaccine trials do not typically evaluate “intra-group dose escalation”, in which individuals would first receive a prime with a low dose and then a boost with a higher dose. Here, we performed studies in mice to determine the immunological effects of intra-group dose escalation. We first primed mice with a low dose or a standard dose of an Ad5-based COVID-19 vaccine, followed by homologous boosting with a standard dose to measure anamnestic immune responses. Our data show that limiting the priming dose of Ad5-vectored vaccines improves the boosting capacity of CD8+ T cell responses and antibody responses. This beneficial effect of limiting the priming dose was associated with reduced generation of vector-specific immune responses, as well as cell-intrinsic differences in memory T cell differentiation.


Adenovirus-based SARS-CoV-2 vaccines are currently being deployed in humans to prevent COVID-19, including the Johnson & Johnson’s Janssen vaccine, AstraZeneca/Oxford vaccine, CanSino vaccine, and the Sputnik V vaccine. These vaccines have already been administered to millions of people, and have shown potent immunogenicity, safety, and efficacy against severe SARS-CoV-2 infection. The CanSino and Sputnik V vaccines specifically utilize Ad5, which is the same vector platform used in our studies. Ad5 is among the most well-studied vaccine vectors, due in part to its extraordinary immunogenicity (2, 26). Although SARS-CoV-2 vaccines prevent severe disease and death, they do not always confer durable sterilizing immunity, warranting the further optimization of current vaccine regimens.
Here, we show that fractionating the priming dose of an adenovirus-based SARS-CoV-2 vaccine confers an unexpected immunological benefit after boosting. Importantly, a possible trade-off of fractionating the priming dose is that it may initially result in lower antigen-specific immune responses, which may render the host transiently more susceptible to infection, although this requires investigation. While it remains unclear what the minimum level of immune responses required to protect against severe COVID-19 is, a recent study demonstrated that even a low dose single-shot of an Ad26-based SARS-CoV-2 vaccine is sufficient to confer protection against severe disease in primates (27). Similarly, a single dose of a DNA-based SARS-CoV-2 vaccine, which is ~1000-fold less immunogenic than a LD single-shot of the Ad5-based vaccine, was able to protect macaques from a SARS-CoV-2 challenge (28). These prior papers suggest that low levels of immune responses are sufficient to protect against severe COVID-19 (at least during the short time frame when the vaccines were tested), which can explain the extraordinary success of multiple SARS-CoV-2 vaccines in the last year.
There are historical examples of vaccine dose fractionation, also known as dose-sparing, as a way to enable more people to get vaccinated. For instance, prior studies have shown that administering a fifth of the recommended dose of the yellow fever virus vaccine results in comparable immune responses relative to the recommended dose, without selection of escape variants (29). Similar results have been reported for influenza, measles, polio, and typhoid vaccines (3032), which has led to discussions of whether SARS-CoV-2 vaccine doses should be fractionated to enable more people to get vaccinated. However, a counterargument against vaccine fractionation is that it may result in suboptimal immune protection (relative to standard dose) before the boost is administered.
What is the possible mechanistic basis for the improved recall response observed in the LD/SD regimen? First, we observed that a LD prime induced lower expression of inhibitory receptors and higher expression of central memory markers on CD8+ T cells relative to a SD prime. Second, anti-vector immunity was substantially lower after a LD prime relative to a SD prime. Lower anti-vector immunity may facilitate transgene expression and antigen presentation, as well as more de novo priming following a subsequent boost. Interestingly, Ad5-specific antibodies did not prevent Ad5 from entering muscle cells, but resulted in more rapid elimination of Ad5 after 24 hours, likely via effector mechanisms. Whether blocking effector functions (such as ADCC) can allow the re-utilization of Ad5 vectors, especially in the context of high pre-existing immunity, requires further investigation. Our data with a simian immunodeficiency virus (SIV) vaccine and an OVA vaccine suggest that our findings may also be useful to improve other Ad5-based vaccines. Nevertheless, it is unclear why only the transgene-specific response (but not the vector-specific response) was increased in the LD/SD regimen. The hexon is the most abundant adenovirus capsid protein and is considered to be the main target for adenovirus neutralizing antibodies (33, 34). Future studies should determine if the improvement of recall responses in the LD primed mice is dependent on the level of antigen expression, and whether similar effects can be observed with other vector platforms different than Ad5.
Conceptually, our findings are counterintuitive to conventional wisdom because we show that when it comes to the immune system, sometimes “less is more.” Antigen is the first signal required for the activation of the adaptive immune system, but here we show an example where a lower amount of priming antigen results in superior recall response later during boosting. We also show that reducing the priming dose of a vaccine favors Tcm differentiation, which is characterized by high expression of the IL-7 receptor α chain (CD127) that promotes long-term T cell survival. Tcm CD8+ T cells also exhibit enhanced recall capacity (14), which may explain the improved secondary expansion that was observed in our LD/SD regimen.
The LD/SD regimen also induced superior antibody responses compared to the SD/SD regimen. Strikingly, the LD/SD regimen elicited a 72-fold improvement in the neutralizing antibody response, compared to the SD/SD regimen. In vitro antibody neutralization is directly correlated with in vivo protection against SARS-CoV-2 challenges (28), but we did not perform in vivo SARS-CoV-2 challenges to compare immune protection, because in our pilot studies in k18-ACE2 mice, even a single-shot of the Ad5-SARS-CoV-2 spike vaccine provided robust protection. Similar efficacy has been described after a single dose with other adenovirus-based SARS-CoV-2 vaccines (3537). It is plausible that a LD/SD regimen may confer a protective advantage over a SD/SD regimen months or years after vaccination when responses wane, which might reduce the need for a third boost. The LD/SD regimen may also be useful in individuals who develop suboptimal immune responses to vaccines, or those who may benefit from having a higher level of immune responses. Moreover, reducing vaccine dose may result in fewer side effects or adverse events, since reactogenicity is directly proportional to vaccine dose (3840). There are concerns that SARS-CoV-1 may re-enter the human population, and an important question is whether the current SARS-CoV-2 vaccines could cross-protect against SARS-CoV-1. We analyzed cross-reactive antibody (binding to SARS-CoV-1 spike), and we show that the LD/SD regimen resulted in higher cross-reactive antibody levels compared to the SD/SD regimen. Notably, the CD8+ T cell response that we measured in these studies was specific for a highly conserved epitope (VNFNFNGL) that is present across multiple coronaviruses, including SARS-CoV-2, SARS-CoV-1, RatG13, HKU3, and WIV1, among others. Altogether, the LD/SD regimen may be particularly effective in the context of universal coronavirus vaccines, because this regimen induces potent levels of cross-reactive antibody and CD8+ T cell responses.
The extent to which our results generalize to humans has not been determined, but a recent clinical trial with an adenovirus-based SARS-CoV-2 vaccine (ChAdOx1, nCoV-19), in which the priming dose was unintentionally reduced to half, reported superior efficacy relative to standard dose (90% efficacy for LD/SD, versus 62% efficacy for SD/SD) (18). However, in those studies it was unclear whether the improvement in the LD/SD group was due to prolonging the prime-boost interval or due to the priming dose itself. Our results bring more clarity to this issue, as we show that the priming dose alone can have a substantial qualitative effect on immune responses in mice. Historically, phase 1 vaccine trials are designed to compare a range of vaccine doses among different groups of individuals. However, they do not typically analyze the effect of dose-escalation within the same individual. The data presented support exploring intra-group vaccine dose escalation in future clinical trials and may be useful to understand the immunological effects of vaccine fractionation.



We thank Drs. Susan Weiss, Stanley Perlman, and Thomas Gallagher for discussions.

FUNDING: This work was possible with a grant from the Emerging and Re-Emerging Pathogens Program (EREPP) at Northwestern University, and a grant from the National Institute on Drug Abuse (NIDA, DP2DA051912) to P.P.M.

AUTHOR CONTRIBUTIONS: P.P.M., S.S., N.P., T.D., and T.C. designed and conducted the experiments. P.P.M. wrote the manuscript with feedback from all authors.

COMPETING INTERESTS: Pablo Penaloza-MacMaster reports being Task Force Advisor to the Illinois Department of Public Health (IDPH) on SARS-CoV-2 vaccine approval and implementation in the state of Illinois. Pablo Penaloza-MacMaster is also member of the Northwestern COVID-19 Vaccine Communication and Evaluation Network (CoVAXCEN) at Northwestern University’s Institute for Global Health.

DATA AND MATERIALS AVAILABILITY: RNA and TCR sequencing data are available in the Gene Expression Omnibus under accession number GSE173567. All other data needed to evaluate the conclusions in the paper and present in the paper or the Supplementary Materials.

This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.

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