Nonviral delivery of self-amplifying RNA vaccines

In the paper

“Although plasmid DNA (pDNA) vaccines have proven to be a flexible platform and are broadly effective in small animal models, they have generally lacked potency in human clinical trials (2).”

What the sources say

• Supported

  The Preclinical studies section reports protective immunity against a broad range of pathogens in small-animal studies and describes applications beyond infectious disease. The DNA vaccine platform: room for improvement section says responses in humans have been significantly weaker than in mice, and Initial DNA vaccine studies in humans says greater potency is needed. (LLM, conf=0.92)

  cites [2] source

In the paper

“However, with increasing life expectancy in high-income countries and newly emerging infections and poverty in low-income countries, new technologies will be required to address changing vaccine needs (1).”

What the sources say

• Supported

  The Main section identifies an aging society, emerging infections, and poverty in low-income countries as changing health needs. It also says emerging infections require appropriate technologies and that research into novel vaccine technologies remains a priority. No page numbers are available in our copy. (LLM, conf=0.92)

  cites [1] source

In the paper

“VRPs are potent vaccines in mice (10,11), nonhuman primates (11,22), and humans (25).”

What the sources say

• Supported

  The Discussion reports potent vaccine responses in mice, and the Abstract states that VEE replicon particles induced potent, protective immune responses in primates. In the citing sentence, this source is cited for mice and nonhuman primates; the human claim is assigned to citation 25, not this paper. (LLM, conf=0.91)

  cites [10] source
• ?Not checked — no full text of the sourcecites [11] source
• Partly supported

  The Discussion (page unavailable) reports complete protection of rhesus macaques given an intramuscular VRP prime plus Env-protein boost, supporting the nonhuman-primate portion attributed to this source. It also says none of the macaques given VRP alone were protected, so the unqualified description of VRPs as potent vaccines overstates this study. The separate mice and humans citations are not being judged here. (LLM, conf=0.85)

  cites [22] sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [25] source

In the paper

“There are limited published data on the in vivo delivery of self-amplifying RNA using nonviral delivery strategies (3,14,18), and none has taken advantage of the recently developed, clinically suitable delivery systems for siRNA (19,20).”

What the sources say

• ?Not checked — no full text of the sourcecites [3] source
• Supported

  This paper is cited for the first clause: examples of in vivo, nonviral delivery of self-amplifying RNA. The Discussion describes delivery of naked Semliki Forest virus RNA replicons and reports immune responses after intradermal delivery with electroporation. The separate claim about siRNA delivery systems is attached to other citations, not this paper. (LLM, conf=0.90)

  cites [14] source
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [18]
• ?Not checked — no full text of the sourcecites [19] source
• Supported

  The Main section (page unavailable) describes synthetic, nonviral siRNA delivery systems that have worked in vivo and says several clinical trials use delivery agents. This supports the cited description of clinically promising siRNA delivery systems; the statement that self-amplifying RNA studies had not used them is the citing authors’ comparison, not a finding attributed to this source. (LLM, conf=0.87)

  cites [20] source

In the paper

“Injection of naked mRNA or self-amplifying RNA in vivo induces gene expression and generates immune responses (4, 11-13), with self-amplifying RNA being more efficient for gene expression in situ (13,14).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites [4]
• ?Not checked — no full text of the sourcecites [11] source
• ?Not checked — no full text of the sourcecites [12] source
• Partly supported

  The in vivo gene-expression results report that injected replicative mRNA produced substantially more luciferase than non-replicative mRNA in muscle. The Discussion reports an immune response from prior CEA mRNA injections, but this study did not establish that its self-amplifying RNA generated an immune response; it discusses that possibility as a hypothesis (page not provided). (LLM, conf=0.87)

  cites [13] sourceExperimental — accuracy not yet measured
• Partly supported

  The Results section ('Relative expression levels and cellular immune response'; page unavailable) reports in vivo gene expression from both mRNA and replicon RNA, with lower, shorter-lived expression from mRNA. Replicon RNA elicited immune responses, but naked mRNA produced no detectable specific immune response in this study's single-injection experiments. The Introduction does report immune responses to mRNA vaccination in other studies, so the broad claim is supported only with that qualification. (LLM, conf=0.86)

  cites [14] sourceExperimental — accuracy not yet measured

In the paper

“The F protein of RSV is a conserved target of neutralizing antibodies and a promising antigen for RSV vaccine development (29).”

What the sources say

• Supported

  The introductory passage following the Abstract explicitly calls RSV F “a conserved target of neutralizing antibodies” and “a promising antigen for RSV candidate vaccines.” The Discussion further reports that an engineered F antigen elicited high neutralizing antibody titers and protected cotton rats from RSV challenge. (LLM, conf=0.95)

  cites [29] source

In the paper

“The neutralization titers elicited by LNP/RNA are above the titer of 380 that correlates with protection in cotton rats (30) and similar to passively acquired serum neutralizing titers that correlate with protection of human infants from severe RSV disease (31,32).”

What the sources say

• ?Not checked — no full text of the sourcecites [30] source
• Supported

  Split into 2 atoms — 2 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [31] source
• ?Not checked — no full text of the sourcecites [32] source

In the paper

“Currently there is no vaccine for RSV and, because of a history of disease enhancement upon natural infection after immunization with an RSV vaccine candidate that elicited T H 2-biased (and non-neutralizing) RSV-specific immune responses in RSV-naive infants, elicitation of a non-T H 2-biased RSV-specific response is considered an essential attribute for infant RSV vaccine candidates (33).”

What the sources say

• Partly supported

  The Major hurdles section says adequate RSV vaccine options were unavailable and describes enhanced disease in infants after the formalin-inactivated vaccine. The Vaccine-enhanced disease section reports poor neutralizing antibody responses in those children, and the Role of T cells in vaccine-enhanced illness section recommends avoiding Th2 responses in RSV vaccines. However, it identifies clear Th2 induction in immunized animal models, not as a directly established response in the infants; their lung eosinophilia is cited as a reason for concern. (LLM, conf=0.86)

  cites [33] sourceExperimental — accuracy not yet measured

In the paper

“The shorter duration of expression in situ after RNA immunization compared with pDNA may be related to the induction of apoptosis of the transfected cell, which could occur during RNA amplification, leading to transient gene expression (12,13).”

What the sources say

• ?Not checked — no full text of the sourcecites [12] source
• Partly supported

  The in vivo results report that replicative mRNA expression returned to baseline within 28 days, while plasmid DNA expression remained above baseline (Augmented gene expression in vivo). The Discussion proposes host-cell protein-synthesis inhibition and resulting cytopathogenicity as one possible explanation for the decline in mRNA-directed expression. It does not specifically identify apoptosis or establish that it occurs during RNA amplification. (LLM, conf=0.85)

  cites [13] sourceExperimental — accuracy not yet measured

In the paper

“Recently, there has been an exponential growth in the development of clinically suitable nonviral delivery systems for siRNA (19,20); we have taken advantage of these innovations to develop the LNP/RNA formulation and have now embarked on a more extensive evaluation of other nonviral delivery systems.”

What the sources say

• ?Not checked — no full text of the sourcecites [19] source
• Partly supported

  The Main section describes advances in synthetic nonviral siRNA carriers and reports that some delivery agents are being used in clinical trials. It does not establish exponential growth, and it says safe, effective delivery remains a barrier rather than characterizing these systems generally as clinically suitable. Page numbers were not provided in our copy. (LLM, conf=0.85)

  cites [20] sourceExperimental — accuracy not yet measured

In the paper

“The same self-amplifying RNA, when delivered using a VRP (1 × 10 6 IU), produced twice the bioluminescence at day 3 compared with LNP/RNA, but expression decayed much more rapidly and reached background levels by day 28 (Fig. 2B).”

In the paper

“EP delivery of pDNA (at a high dose of 20 μg) elicited anti-F IgG titers (GMTs of 6,712) on average 3.9-fold less than those elicited by 0.1 μg of LNP/RNA, although not statistically different.”

In the paper

“On the other hand, mice administered a pDNA-encoding luciferase (10 μg) delivered using EP in situ displayed the highest measurable bioluminescence at all time points tested, and this level remained high for at least 63 d (Fig. 2D).”

In the paper

“Encapsulation of a 10-fold lower dose of RNA (0.1 μg) in LNPs increased the serum SEAP concentration and reduced the variability between animals.”

In the paper

“Six days after intramuscular (i.m.) injection of 1 μg naked self-amplifying RNA encoding secreted alkaline phosphatase (SEAP), gene expression, as determined by serum SEAP concentration, was measurable but highly variable (Fig. 1D).”

In the paper

“This contrasted with what was observed for naked RNA, which required two vaccinations and microgram doses of RNA.”

In the paper

“The LNP/RNA was statistically superior to naked RNA and equivalent to the 5 ×10 6 IU of VRPs [GMTs 5,861 (F-specific IgG) and 1,690 (neutralization)].”

In the paper

“Addition of empty LNPs (without RNA) to naked RNA (0.1 μg) decreased the serum SEAP concentration relative to LNP-encapsulated RNA, indicating that RNA encapsulation was necessary for efficient RNA delivery and reporter gene expression.”

In the paper

“A recombinant F protein subunit vaccine formulated with alum was shown to be the most potent vaccine for induction of total and protective antibody responses.”

In the paper

“Bioluminescence intensity induced by LNP/RNA was substantially greater than naked RNA, pDNA, and pDNA launched RNA, and was comparable to the bioluminescence produced by 1 × 10 6 infectious units (IU) of VRPs.”

In the paper

“Agarose gel electrophoresis showed that RNA integrity was maintained during formulation (Fig. 1C, lane 4) and that the LNPs protected the RNA from degradation by RNase A (Fig. 1C, lane 5).”

In the paper

“In an extended study (Fig. 2 B-D), mice injected with LNP/RNA (1 μg) showed measurable bioluminescence that was already high on day 3, peaked on day 7, and decreased to background by day 63 (Fig. 2C).”

In the paper

“The potency of this unique LNP/RNA vaccine in mice and cotton rats was comparable to a single-cycle alphavirus vector (VRP, 1 × 10 6 IU) at a reasonable dose of RNA (1 μg) and was generally comparable to pDNA delivered using EP at higher doses.”

In the paper

“All self-amplifying RNA vaccines provided protection from a nasal RSV challenge, reducing the lung viral load >1,000-fold compared with control animals (Fig. 5C) and these data are in agreement with previous reports for naked RNA (12).”

What the sources say

• ?Not checked — no full text of the sourcecites [12] source

In the paper

“The LNP/RNA formulation boosted F-specific IgG titers approximately 8-fold (GMTs 4,355) and neutralization titers 10-fold (GMTs 1,493) relative to the same dose of naked RNA [GMTs of 526 (F-specific IgG) and 154 (neutralization)].”

In the paper

“An ethanol dilution process (28) was used to produce small uniform lipid particles with a high RNA encapsulation efficiency.”

What the sources say

• ?Not checked — no full text of the sourcecites [28] source

In the paper

“The number-weighted mean diameters ranged from 79 to 121 nm and Z-average diameters from 130 to 164 nm with a polydispersity index from 0.09 to 0.14.”

In the paper

“The ratio of F-specific IgG titer to RSV neutralization titer (Table S2) is low for the LNP/ RNA vaccine relative to the RSV-F subunit adsorbed on alum; thus it may be particularly suitable for development into a safe and effective infant RSV vaccine candidate.”

In the paper

“The LNP/RNA provides this benefit (Fig. 4) in addition to strong cellular immune responses.”

In the paper

“(iv) the generation of a humoral and cellular immune responses (both CD4 and CD8).”

In the paper

“Facilitated delivery of RNA, either by VRPs or LNPs, resulted in higher levels of reporter gene expression (SEAP and luciferase) than naked RNA delivery and considerably lower variability of expression between animals.”

In the paper

“However, many of the obstacles to mRNA vaccine development have been surmounted, and recently there has been a revival in the use of nonamplifying mRNA vaccines for cancer (6), allergy (7), and gene therapy (8).”

What the sources say

• ?Not checked — no full text of the sourcecites [6] source
• ?Not checked — no full text of the sourcecites [7] source
• ?Not checked — no full text of the sourcecites [8] source

In the paper

“Naturally transient and cytosolically active mRNA can now be produced at sufficient quantity and quality for human clinical trials (6) and is seen by many (9) as a safer and more potent alternative to pDNA for vaccination.”

What the sources say

• ?Not checked — no full text of the sourcecites [6] source
• ?Not checked — no full text of the sourcecites [9] source

In the paper

“Viral vector-based technologies are generally regarded as the most efficient means to deliver nucleic acids into cells, but their utility can be restricted by preexisting or vaccine-induced antivector immunity that can decrease vaccine potency (12).”

What the sources say

• ?Not checked — no full text of the sourcecites [12] source

In the paper

“Recombinant viral vector technologies have the advantage of efficient delivery of the nucleic acid payload, but their utility is often hampered by antivector immunity, production limitations, and safety concerns (3,4).”

What the sources say

• ?Not checked — no full text of the sourcecites [3] source
• ?Not checked — no full text of the sourcecites [4]

In the paper

“Various RNA vaccines have been evaluated in human clinical trials, including naked and formulated non-amplifying mRNA (37-39) and self-amplifying RNA packaged in VRPs (25).”

What the sources say

• ?Not checked — no full text of the sourcecites [37] source
• ?Not checked — no full text of the sourcecites [38] source
• ?Not checked — no full text of the sourcecites [39] source
• ?Not checked — no full text of the sourcecites [25] source

In the paper

“RNA vaccines avoid this rate-limiting step by using cytoplasmic amplification and expression, resulting in more efficient transfection of different cell types, including quiescent or slowly proliferating cells, such as vascular endothelia or myocytes.”

In the paper

“Therefore, the LNP formulation markedly enhances the potency of the self-amplifying RNA, thereby generating a robust and potentially generic vaccine technology.”

In the paper

“Image acquisition times were kept constant as bioluminescence was measured with a cooled CCD camera.”

In the paper

“Those studies showed RNA vaccines to be well tolerated and immunogenic.”

What the sources say

• ?Not checked — no full text of the sourcecites [37] source
• ?Not checked — no full text of the sourcecites [38] source
• ?Not checked — no full text of the sourcecites [39] source
• ?Not checked — no full text of the sourcecites [25] source

In the paper

“In addition, because the replication cycle of the self-amplifying RNA is strictly cytoplasmic, the need for codon modification is diminished, and the risks of splicing and destruction of the transcript are avoided.”

In the paper

“For an LNP/RNA vaccine, this sequestration might result in antigen production and/or stimulation of innate immune pathways within these immune cells.”

In the paper

“(ii) elimination of theoretical risks of generating infectious virus through recombination during production”

In the paper

“The LNP delivery system may achieve these effects by a combination of increased transfection efficiency and protection of the RNA from enzymatic degradation at the site of injection.”